Multi-antigenic alphavirus replicon particles and methods

ABSTRACT

Viral replicon selected nucleic acid expression libraries are useful for analyzing multiple antigens associated with a parasite, pathogen or neoplasia or for preparing immunogenic compositions for generating immune responses specific for the parasite, pathogen or neoplasia. Alphavirus replicon particles representative of the nucleic acid expression library are preferred. The nucleic acid library can be a random library, or it can be prepared after a selection step, for example, by differential hybridization prior to cloning into the replicon vector.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional ApplicationNos. 60/433,299 and 60/433,058, both filed Dec. 13, 2002.

ACKNOWLEDGMENT OF FEDERAL RESEARCH SUPPORT

[0002] Not applicable.

BACKGROUND OF THE INVENTION

[0003] The present invention relates to recombinant DNA technology, andin particular to introducing foreign nucleic acid(s) into a eukaryotichost cell, and more particularly to producing infective,propagation-defective virus-like particles which collectively direct theexpression of a representative set of immunogenic proteins (anexpression library) of a pathogen (virus, fungus, bacterium orprotozoan), parasite or tumor cell. These libraries have applications inhuman and veterinary medicine.

[0004] A vaccine is one of the most efficacious, safe and economicalstrategies for preventing disease and controlling the spread of disease.Conventional vaccines are a form of immunoprophylaxis given beforedisease occurrence to afford immunoprotection by generating a stronghost immunological memory against a specific antigen. The primary aim ofvaccination is to activate the adaptive specific immune response,primarily to generate B and T lymphocytes against specific antigen(s)associated with the disease or the disease agent.

[0005] Similarly, cancer vaccines aim to generate immune responsesagainst cancer tumor-associated antigens. Cancers can be immunogenic andcan activate host immune responses capable of controlling the diseaseand causing tumor regression. However, cancer at the same time can bespecifically and nonspecifically immunosuppressive and can evade thehost's immune system. Many protein/glycoprotein tumor-associatedantigens have been identified and linked to certain types of cancer.Her-2-neu, PSA, PSMA, MAGE-3, MAGE-1, gp100, TRP-2, tyrosinase, MART-1,β-HCG, CEA, Ras; B-catenin, gp43, GAGE-1, BAGE-1, MUC-1,2,3, and HSP-70are just a few examples.

[0006] Multiple approaches are being assessed in immunizing cancerpatients with tumor-associated antigens (TAAs). Vaccines in clinical usefall into several categories determined by their components, which rangefrom whole cells to immunogenic peptides. Whole cell and cell lysatevaccines can be autologous or allogeneic vaccines, depending on the hostorigin of the cancer cells. An autologous whole cell cancer vaccine is apatient-specific formulation made from the patient's own tumor. To date,many autologous cancer vaccines have not been clinically successfulunless they are modified to increase their intrinsic immunogenicity, forexample by the co-expression of lymphokines such as GM-CSF (Ward et.al., 2002. Cancer Immunol. Immunother. 51:351-7). Because they arepatient-specific, they can also be costly and limited to those patientsfrom whom cancer cells can be obtained in sufficient quantity to producea single-cell suspension. In addition, the inherently limited number ofcells is problematic with respect to the need for modification or formultiple vaccinations, making an autologous formulation impractical forprophylaxis or treatment of early disease. Some of these problems aresolved with allogeneic whole cell vaccines or genetically engineeredwhole cell vaccines where instead of supplying immunostimulatory agentssuch as lymphokines exogenously with the tumor vaccine, the tumor cellsare genetically modified to express the lymphokine endogenously.However, these methods may be time consuming and prohibitively expensiveto produce.

[0007] Natural and recombinant cancer protein antigen vaccines aresubunit vaccines. Unlike whole cell vaccines, these subunit vaccinescontain defined immunogenic antigens at standardized levels. The keyproblem with such vaccines is finding the right adjuvant and deliverysystem. In addition, purification of natural or recombinant tumorantigens is tedious and not always logistically practical. Proteincancer vaccines require culturing tumor cells, purifying tumor antigens,or producing specific peptides or recombinant proteins. In addition,vaccines that are made solely from tumor protein/peptides pose intrinsicproblems in that they can be limited in the ability to be directed intothe correct antigen presentation pathways or may not be recognized bythe host due to host major histocompatibility complex (MHC)polymorphisms. For these reasons, whole cell, or vector delivered tumorvaccines expressing a large array of tumor antigens are anticipated tobe preferred vaccination methods. Vaccines which include nucleic acidencoding the tumor antigens rather than vaccines comprising the antigenitself, address some of these problems. To date these approaches haveshown the most promise in pre-clinical and clinical testing. Amongst thecurrent technologies being applied to cancer vaccination, two particularsystems have shown significant potential for application in this field.The first is delivery of TAAs using viral vectors, including but notlimited to adenoviral, adeno associated virus, retroviral, poxviruses,flaviviruses, picornaviruses, herpesviruses and alphaviruses (see WO99/51263). The second is vaccination with tumor cell protein or RNAusing ex vivo derived dendritic cells as the delivery vehicle fortransfer and expression of the TAAs into the host (Heiser et al., 2002.J. Clin. Inv. 109:409-417 and Kumamoto et al., 2002. Nature Biotech.20:64-69).

[0008] A limiting factor in many tumor vaccine approaches appears to bethe limited availability of known tumor-specific antigens. Thesetumor-specific antigens can vary not only between tissue type from whichthe tumor originated, but may even vary from cell-to-cell within thesame tumor. A confounding problem associated with using only a limitednumber of tumor antigen targets in a vaccine is the potential for “tumorescape” where the tumor essentially evades detection by the vaccineinduced immune effector cells by deleting certain tumor associatedantigens.

[0009] This observation prompted investigators to design cancer vaccinesexpressing multiple antigens to reduce the propensity of tumor escape.Unfortunately due to the limited number of antigens that have beenidentified to date, this is not a feasible approach for the majority oftumors. Therefore, a more recent evolution of cancer therapy has beenthe use of entire tumor antigen libraries. This combines multiplebeneficial characteristics one would want in a cancer vaccine. A vaccineencoding an entire tumor antigen repertoire negates the need for antigenidentification and isolation; essentially the vaccine recipient's immunesystem is allowed to make this choice in determining which TAAs theindividual will respond to. The second distinct advantage of thisapproach is that, since the repertoire of antigens being expressed is sobroad, the chance of tumor escape is minimized or eliminated entirely.Currently this approach is most actively being pursued using dendriticcells to deliver tumor antigen libraries. These cells, which function asantigen presenting cells by presenting the tumor antigens to the immunesystem, are isolated from each cancer patient, cultured and expanded invitro, loaded with tumor antigen either in the form of protein ornucleic acid; see U.S. Pat. Nos. 5,853,719 and 6,306,388. This approachhas generated promising clinical data in human testing and has shown theability to retard tumor growth in some individuals, and even to drivetumor regression in a number of patients (Sadanaga et al., 2001, Clin.Cancer Res. 7:2277-84). The major drawback for this technology is theneed for in vitro culture, expansion and antigen loading of the patientderived dendritic cells prior to vaccination of each individual. This isa time consuming and expensive process, and can be highly variable sincethe dendritic cell population from individual to individual can varywidely in its phenotype, growth characteristics and activity.

[0010] To date, naked DNA, RNA, viral and bacterial vectors have beentested for their ability to induce cancer specific responses against atumor antigen library. An alternative approach is the use of viralvectors to deliver a tumor antigen library to a cancer patient. To date,some success has been achieved with naked nucleic acid expressionlibraries; e.g., see U.S. Pat. Nos. 5,989,553 and 5,703,057. Attempts toaugment the immune responses elicited to naked nucleic acid vectorsinclude the use of self-replicating viral vectors delivered in the formof naked RNA or DNA (Ying et al., 1999, Nature Medicine, 5:823-827).

[0011] Viral vectors have shown great promise in pre-clinical andclinical testing for prevention of a number of infectious diseasetargets. One of the most pressing issues for development of viralvectors for prophylactic and therapeutic vaccine uses in humans is theability to produce enough particles in a regulatory acceptable form. Formany viral systems, this goal is within reach and a number of vectorsystems have produced positive immune response and safety profiles inclinical trials. However, most production schemes for vaccine vectorplatforms are focused on production of large quantities of vaccineparticles expressing single or at the most two or three known antigensfor specific disease targets e.g. the gag, pol and env genes of HIV inpoxvirus vectors. However, in most cases, these large-scalemanufacturing approaches are not practical for the manufacture ofindividual patient-specific vaccines.

[0012] Alphaviral vector delivery systems have been identified asattractive vaccine vectors for a number of reasons including: highexpression of heterologous gene sequences, the derivation ofnon-replicating (alpha)virus replicon particles (ARP) with good safetyprofiles, an RNA genome which replicates in the cytoplasm of the targetcell and negates the chance of genomic integration of the vector, andfinally the demonstration that certain alphaviral vectors areintrinsically targeted for replication in dendritic cells and thus cangenerate strong and comprehensive immune responses to a multitude ofvaccine antigens (reviewed in Rayner, Dryga and Kamrud, 2002, Rev. Med.Virol. 12:279-296). The Alphavirus genus includes a variety of viruses,all of which are members of the Togaviridae family. The alphavirusesinclude Eastern Equine Encephalitis Virus (EEE), Venezuelan EquineEncephalitis Virus (VEE), Everglades Virus, Mucambo Virus, Pixuna Virus,Western Equine Encephalitis Virus (WEE), Sindbis Virus, Semliki ForestVirus, Middleburg Virus, Chikungunya Virus, O'nyong-nyong Virus, RossRiver Virus, Barmah Forest Virus, Getah Virus, Sagiyama Virus, BebaruVirus, Mayaro Virus, Una Virus, Aura Virus, Whataroa Virus, BabankiVirus, Kyzylagach Virus, Highlands J Virus, Fort Morgan Virus, NdumuVirus, and Buggy Creek Virus. The viral genome is a single-stranded,messenger-sense RNA, modified at the 5′-end with a methylated cap and atthe 3′-end with a variable-length poly (A) tract. Structural subunitscontaining a single viral protein, C, associated with the RNA genome inan icosahedral nucleocapsid. In the virion, the capsid is surrounded bya lipid envelope covered with a regular array of transmembrane proteinspikes, each of which consists of a heterodimeric complex of twoglycoproteins, usually E1 and E2. See Pedersen et al., J. Virol 14:40(1974). The Sindbis and Semliki Forest viruses are considered theprototypical alphaviruses and have been studied extensively. SeeSchlesinger, The Togaviridae and Flaviviridae, Plenum Publishing Corp.,N.Y. (1986). The VEE virus has also been extensively studied. See, e.g.,U.S. Pat. No. 5,185,440, and other references cited herein.

[0013] The studies of these viruses have led to the development oftechniques for vaccination against the alphavirus diseases and againstother diseases through the use of alphavirus vectors for theintroduction of foreign DNA encoding antigens of interest. See U.S. Pat.No. 5,185,440 to Davis et al., and PCT Publication WO 92/10578. Theintroduction of foreign expressible DNA into eukaryotic cells has becomea topic of increasing interest. It is well known that live, attenuatedviral vaccines are among the most successful means of controlling viraldisease. However, for some viral (or other) pathogens, immunization witha live virus strain may be either impractical or unsafe. One alternativestrategy is the insertion of sequences encoding immunizing antigens ofsuch agents into a live, replicating strain of another virus. One suchsystem utilizing a live VEE vector is described in U.S. Pat. No.5,505,947 to Johnston et al. Another such system is described by Hahn etal., 1992, Proc. Natl. Acad. Sci. USA 89:2679-2683, wherein Sindbisvirus constructs express a truncated form of the influenza hemagglutininprotein. Another approach is the use of infective, propagation-defectivealphavirus particles, as described in U.S. Pat. No. 6,190,666 to Garoffet al., U.S. Pat. Nos. 5,792,462 and 6,156,558 to Johnston et al., U.S.Published Application No. 2002/0015945 A1 (Polo et al.), U.S. PublishedApplication No. 2001/0016199 (Johnston et al.), Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377 and Pushko et al. (1997)Virology 239:389-401. Alphaviruses have also been shown to be relativelyeasy to genetically manipulate, as reflected by a number of applicationsusing alphaviruses as genomic expression libraries, e.g., see U.S. Pat.No. 6,197,502. The use of Semliki Forest Virus (SFV) vectors expressinga library of antigens has also been explored in animal models where SFVparticles expressing a library of tumor antigens were used to infectdendritic cells in vitro and the dendritic cells were used to immunizemice showing some protection in a glioma model (Yamanaka et al., 2001,J. Neurosurg. 94:474-81).

[0014] There is a longfelt need in the art for nucleic acid sequencesencoding foreign antigens which can be used to immunize a person or ananimal against neoplastic conditions or against parasite or pathogeninfection, especially where there is no attenuated strain or where theneoplasia, parasite or pathogen is not well characterized at themolecular level, or where it is recognized that protective immunizationrequires the expression of multiple antigens.

SUMMARY OF THE INVENTION

[0015] It is an object of the present invention to provide a virusreplicon particle preparation derived from a neoplastic cell, pathogenor a parasite and immunogenic compositions comprising same. Thepreparation contains a multiplicity of expressible coding sequencesderived from the neoplastic cell, pathogen or parasite, and expressionof the coding sequences in a human or animal patient to whom thepreparation is administered results in the generation of an immuneresponse to the multiplicity of antigenic determinants encoded by andexpressed from the alphavirus replicon nucleic acid. The immunogeniccomposition comprises the alphavirus replicon particle preparation ofinterest and a pharmaceutically acceptable carrier, and advantageouslyfurther comprises an immunological adjuvant and/or a cytokine to improveor stimulate the immune response. The alphavirus replicon can be anyalphavirus replicon RNA vector derived from VEE, Sindbis virus, SouthAfrican Arbovirus No. 86, Semliki Forest virus, among others. Inpreferred embodiments, the alphavirus vector contains one or moreattenuating mutations. Suitable mutations, as well as methods toidentify them, have been described (see, for example, U.S. Pat. Nos.5,505,947; 5,639,650; 5,811,407).

[0016] Routes of administration can include subcutaneous (s.c.),intraperitoneal (i.p.), intramuscular (i.m.), intradermal (i.d.),intravenous (i.v.), intratumoral, intracerebral (i.c.), direct lymphnode inoculation (i.n.), and mucosal routes such as nasal, bronchial,intrarectal, intravaginal and oral routes. Intramuscular administrationis advantageous.

[0017] Dosages in humans and animals can range from about 1×10⁴ to about1×10¹⁰, advantageously at a dose of about 1×10⁶ to about 1×10⁸ per dose.For the vaccine-type immunogenic approaches, the present inventorscontemplate weekly, biweekly or monthly doses for a period of about 1 toabout 12 months, or longer. This can be followed by boostervaccinations, on an as needed basis, e.g. annually.

[0018] Especially in the case where the alphavirus replicon preparationis derived from tumor cells from a specific patient, a patient specificvaccine preparation is made and administered back to the sameindividual; i.e. the autologous vaccine approach. Also within the scopeof the present invention is an allogeneic approach, in which the viralreplicon population derived from one patient's tumor cells isadministered to another patient suffering from, believed to be sufferingfrom or at high risk for the same neoplastic condition. An example of ahigh risk patient is an individual with a genetic predisposition orproven hereditary increased risk for cancer. For example, breast canceris associated with high familial risk in female family members ofpatients suffering from breast cancer. Similarly, one might vaccinate anHIV positive individual and at the same time, prophylactically vaccinatetheir non-infected partner with the same vaccine preparation to try toprevent the uninfected individual from becoming infected.

[0019] The present invention further encompasses following the immuneresponses elicited by administration of a virus replicon preparation oran immunogenic composition comprising the same in a patient to identifythose tumor antigens to which the patient has responded. These responsescan be humoral and/or cellular. This approach allows the identificationof novel antigens and enables the use of a more defined population ofantigens with which to immunize the patient. This can be accomplished byadministering boosts with more limited ARP preparations or by carryingout subsequent immunizations of other patients or individuals (in aprophylactic regimen) with the more defined set of antigen-encodingARP-containing immunogenic preparations.

[0020] The present invention also relates to the treatment and/orprevention of infectious diseases and parasite infestations. Using HIVas an example, a successful multi-antigenic HIV ARP vaccine derived froma patient-specific HIV gene or genes directly from an individual's ownviral population can be applied to persons infected with a similargenetic strain of virus or persons exposed, likely to be exposed orpotentially exposed to a similar strain. Particularly, immunogenic ornovel immunogens from the pathogen or parasite of interest can beidentified using the ARPs as a tool to identify new immunogenicproteins. Similarly, multiple strains of a disease causing virus (suchas the recognized clades of HIV) or parasite can be combined into theARP preparation of this invention to provide robust, immunogeniccompositions which are not strain-specific. For example, severaldifferent clades of HIV have been recognized, and they can be combinedto provide a multi-clade HIV vaccine.

[0021] In the case of cancer patients, the administration of ARPscarrying expressible cancer cell antigenic determinants' codingsequences is advantageously accompanied by chemotherapeutic treatments,especially where chemotherapeutic treatments do not ablate the abilityof the immune system to respond to antigens expressed after theadministration of immunogenic compositions comprising the ARPs of thepresent invention.

[0022] The ARP preparations of the present invention, expressingantigens characteristic of a particular type of tumor or cancer, avirus, a bacterial, fungal or protozoan pathogen or a parasite can beadministered in prophylactic or therapeutic treatment regimens, andadministration of the ARPs can be carried out in combination with otherimmunogenic preparations for priming and/or boosting, for example, usingan ARP vaccine prime and dendritic cell vaccine boost, or an ARP primeand an adenoviral vector boost. All possible combinations of DNA, RNA,adenoviruses, picornaviruses, adeno-associated viruses, poxviruses,retroviruses, aphthoviruses, nodaviruses, flaviviruses, dendritic cell,peptides, heat shock proteins, minigenes, whole tumor cells and tumorcell lysate vaccines can be used in conjunction with the ARPs expressinga multiplicity of antigens of interest of the present invention.Adjuvants such as cytokines or chemokines, or ARPs which direct theexpression of chemokines or cytokines, can be utilized in thepreparations of the present invention. The addition of heterologousprime/boosts in combination with the ARP expressing a multiplicity ofgenes would likely be with vector replicons or sets of vector repliconsexpressing single or a relatively small number of tumor antigens. Thisfunctions so as to focus the immune system on specific antigensfollowing or prior to a broader immune response elicited by the ARP(s).Similar such heterologous delivery systems may be used in combinationwith the present alphavirus replicon expression libraries to enhanceand/or maintain addition memory and longterm immune functions.

[0023] A further object of the present invention is the administrationof the ARP-containing immunogenic compositions of the present inventionto a human not only to treat cancer or other pathological states in atherapeutic setting when the patient is positive for tumor, pathogen orparasite, but also once treatment is successful and the patient is inremission. Such ongoing periodic (booster) immunization can facilitatemaintenance of a tumor-free, disease-free or parasite-free state andprevent regression or recurrence of the tumor or disease, respectively.

[0024] A further object of the present invention is the administrationof the ARP-containing immunogenic compositions of the present inventionto an animal (e.g. horse, pig, cow, goat, primate, rabbit, mouse,hamster, avian) to generate immune responses, such as antibodies. Seraor cells collected from such animals are useful in providing polyclonalsera or cells for the production of hybridomas that generate monoclonalsera, such antibody preparations being useful in research, diagnosticand therapeutic applications.

[0025] A further object of the invention is a method for preparingalphaviral replicon particles (ARPs) which collectively encode amultiplicity of antigens from a tumor, a tumor cell, pathogen orparasite. The method includes the steps of preparing DNA or cDNA fromthe tumor, a tumor cell, pathogen or parasite of interest and cloninginto the virus/alphavirus replicon nucleic acid to produce a modifiedvirus/alphavirus replicon nucleic acid, introducing the modifiedviral/alphaviral replicon nucleic acid into a permissive cell, saidmodified viral/alphaviral replicon nucleic acid containing at least avirus packaging signal to produce a modified permissive cell, culturingthe modified permissive cell under conditions allowing expression of atleast one helper function and allowing replication of said modifiedviral/alphaviral nucleic acid and packaging to form ARPs, and desirablycontacting the cultured permissive cells with a Release Medium torelease cell- and debris-bound ARPs. The modified viral/alphaviralreplicon nucleic acid can be introduced into permissive cells whichalready contain and express coding sequences required for packaging, orone or more “helper” DNA or RNA molecules carrying packaging genes canbe introduced together with the modified viral/alphaviral repliconnucleic acid. Optionally, the Release Medium step can be preceded by awash step which does not result in the release of the ARPs from thecells. Advantageously the wash step includes DNase treatment, or DNA canbe digested in an ARP preparation with DNase. DNase, for example, fromSerratia marcescens, can be used at a concentration from 10-1000 unitsper mL, with incubation from 10 to 60 minutes at 37° . The ReleaseMedium is an aqueous medium which desirably is from about pH 6 to 9,desirably from about 6.5 to about 8.5, and contains from about 0.2 toabout 5 M of a salt including but not limited to ammonium acetate,ammonium chloride, sodium chloride, magnesium chloride, calciumchloride, potassium chloride, ammonium sulfate and sodium bicarbonate.It is advantageous that when modified alphaviral replicon nucleic acidsare introduced into the permissive cells by electroporation, the cellsare present in a density of from about 10⁷ to about 5×10⁸ per mL ofelectroporation mixture.

[0026] Advantageously, the cells in which the ARPs are to be producedare synchronized in the G2/M phase of the cell cycle prior toelectroporation with the alphavirus replicon vector and helper nucleicacid(s). Without wishing to be bound by any particular theory, it isbelieved that greater electroporation efficiency and transfer of nucleicacid to the nucleus (in those embodiments of the invention that involvenuclear activity) of the electroporated cell is achieved in such G2/Mphase cells.

BRIEF DESCRIPTION OF THE DRAWING

[0027]FIG. 1 is a bar graph depicting antigen-specific immune responsesin animals vaccinated with multi-antigenic ARP. Antigen-specific immuneresponses (in the form of humoral immunity) as measured by either ELISAand presented as reciprocal geometric mean titer, or Western blot or IFAand presented as the lowest dilution at which antigen specific signalwas detectable. Antigen specific immune responses in the form ofcellular immunity as measured by ELISPOT detection of IFN-γ secretingcells and presented as antigen specific IFN-γ secreting lymphocytes per10⁶ lymphocytes. Animals which received the multi-antigenic ARPpreparation either by a subcutaneous (s.c.) or an intraperitoneal (i.p.)route of inoculation mounted immune responses to all antigens in thepreparation. As a positive control, one group received HIV-Gag ARP andmounted immune responses only specific for Gag. Negative control animalshad no detectable response to any antigen.

DETAILED DESCRIPTION OF THE INVENTION

[0028] In the context of the present application, nm means nanometer, mLmeans milliliter, μL means microliter, pfu/mL means plaque formingunits/milliliter, iu means infectious units, VEE means Venezuelan EquineEncephalitis virus, EMC means Encephalocomyocarditis virus, BHK meansbaby hamster kidney cells, HA means hemagglutinin gene, CAT meanschloramphenicol acetyl transferase, β-gal means β-galactosidase, GFPmeans green fluorescent protein gene, N means nucleocapsid, FACS meansfluorescence activated cell sorter, ELISA means enzyme-linkedimmunosorbent assay, and IRES means internal ribosome entry site. Theexpression “E2 amino acid (e.g., Lys, Thr, etc.) number” indicatesdesignated amino acid at the designated residue of the E2 gene, and isalso used to refer to amino acids at specific residues in the E1 gene.

[0029] The term “alphavirus” has its conventional meaning in the art,and includes the various species of alphaviruses such as Eastern EquineEncephalitis Virus (EEE), Venezuelan Equine Encephalitis Virus (VEE),Everglades Virus, Mucambo Virus, Pixuna Virus, Western EquineEncephalitis Virus (WEE), Sindbis Virus, South African Arbovirus No. 86,Semliki Forest Virus, Middleburg Virus, Chikungunya Virus, O'nyong-nyongVirus, Ross River Virus, Barmah Forest Virus, Getah Virus, SagiyamaVirus, Bebaru Virus, Mayaro Virus, Una Virus, Aura Virus, WhataroaVirus, Babanki Virus, Kyzylagach Virus, Highlands J Virus, Fort MorganVirus, Ndumu Virus, and Buggy Creek Virus. The preferred alphavirus RNAtranscripts for use in the present invention include VEE Virus, SindbisVirus, South African Arbovirus No. 86, and Semliki Forest Virus RNAtranscripts.

[0030] Alphavirus-permissive cells employed in the methods of thepresent invention are cells which, upon transfection with an alphaviralRNA transcript, are capable of producing viral particles. Alphaviruseshave a broad host range. Examples of suitable host cells include, butare not limited to Vero, baby hamster kidney (BHK), DF1, CHO, 293, 293T,chicken embryo fibroblast and insect cells such as SF21, Spodopterafrugiperda; C6/36, Aedes albopictus; TRA-171, Toxorhynchitesamboinensis; RML-12, Aedes aegypti; AP-61, Aedes pseudoscutellaris; andMOS-55, Anopheles gambiae cells.

[0031] The phrases “structural protein” or “alphavirus structuralprotein” as used herein refer to the virally encoded proteins which arerequired for encapsidation of the RNA replicon into a replicon particle,and include the capsid protein, E1 glycoprotein, and E2 glycoprotein. Asdescribed herein, the structural proteins of the alphavirus aredistributed among one or more helper nucleic acids. For example, a firsthelper RNA and a second helper RNA can be used, or a single DNA helperencoding all alphavirus structural proteins, can be used. In additionone or more structural proteins may be located on the same RNA moleculeas the replicon RNA, provided that at least one structural protein isdeleted from the replicon RNA such that the replicon and resultingalphavirus particle are propagation-defective. As used herein, the terms“deleted” or “deletion” mean either total deletion of the specifiedsegment or the deletion of a sufficient portion of the specified segmentto render the segment inoperative or nonfunctional, in accordance withstandard usage. See, e.g., U.S. Pat. No. 4,650,764 to Temin et al. Theterm “replication defective” as used herein is synonymous with“propagation-defective”, and means that the particles produced in agiven host cell cannot produce progeny particles in the other host cell,due to the absence of the helper function, i.e. the alphavirusstructural proteins required for packaging the replicon nucleic acid.However, the replicon nucleic acid is capable of replicating itself andbeing expressed within the host cell into which it has been introduced.

[0032] The helper cell, also referred to as a packaging cell, used toproduce the infectious, propagation defective alphavirus particles, mustexpress or be capable of expressing alphavirus structural proteinssufficient to package the replicon nucleic acid. The structural proteinscan be produced from a set of RNAs, typically two, that are introducedinto the helper cell concomitantly with or prior to introduction of thereplicon vector. The first helper RNA includes RNA encoding at least onealphavirus structural protein but does not encode all alphavirusstructural proteins. The first helper RNA may comprise RNA encoding thealphavirus E1 glycoprotein, but not encoding the alphavirus capsidprotein and the alphavirus E2 glycoprotein. Alternatively, the firsthelper RNA may comprise RNA encoding the alphavirus E2 glycoprotein, butnot encoding the alphavirus capsid protein and the alphavirus E1glycoprotein. In a further embodiment, the first helper RNA may compriseRNA encoding the alphavirus E1 glycoprotein and the alphavirus E2glycoprotein, but not the alphavirus capsid protein. In a fourthembodiment, the first helper RNA may comprise RNA encoding thealphavirus capsid, but none of the alphavirus glycoproteins. In a fifthembodiment, the first helper RNA may comprise RNA encoding the capsidand one of the glycoproteins, i.e. either E1 or E2, but not both.

[0033] In combination with any one of these first helper RNAs, thesecond helper RNA encodes at least one alphavirus structural protein notencoded by the first helper RNA. For example, where the first helper RNAencodes only the alphavirus E1 glycoprotein, the second helper RNA mayencode one or both of the alphavirus capsid protein and the alphavirusE2 glycoprotein. Where the first helper RNA encodes only the alphaviruscapsid protein, the second helper RNA may include RNA encoding one orboth of the alphavirus glycoproteins. Where the first helper RNA encodesonly the alphavirus E2 glycoprotein, the second helper RNA may encodeone or both of the alphavirus capsid protein and the alphavirus E1glycoprotein. Where the first helper RNA encodes both the capsid andalphavirus E1 glycoprotein, the second helper RNA may include RNAencoding one or both of the alphavirus capsid protein and the alphavirusE2 glycoprotein.

[0034] In all of the helper nucleic acids, it is understood that thesemolecules further comprise sequences necessary for expression(encompassing translation and where appropriate, transcription orreplication signals) of the encoded structural protein sequences in thehelper cells. Such sequences can include, for example, promoters (eitherviral, prokaryotic or eukaryotic, inducible or constitutive) and 5′ and3′ viral replicase recognition sequences. In the case of the helpernucleic acids expressing one or more glycoproteins, it is understoodfrom the art that these sequences are advantageously expressed with aleader or signal sequence at the N-terminus of the structural proteincoding region in the nucleic acid constructs. The leader or signalsequence can be derived from the alphavirus, for example E3 or 6k, or itcan be a heterologous sequence such as a tissue plasminogen activatorsignal peptide or a synthetic sequence. Thus, as an example, a firsthelper nucleic acid may be an RNA molecule encoding capsid-E3-E1, andthe second helper nucleic acid may be an RNA molecule encodingcapsid-E3-E2. Alternatively, the first helper RNA can encode capsidalone, and the second helper RNA can encode E3-E2-6k-E1. Additionally,the packaging signal or “encapsidation sequence” that is present in theviral genome is not present in all of the helper nucleic acids.Preferably, the packaging signal is deleted from all of the helpernucleic acids.

[0035] These RNA helpers can be introduced into the cells in a number ofways. They can be expressed from one or more expression cassettes thathave been stably transformed into the cells, thereby establishingpackaging cell lines (see, for example, U.S. Pat. No. 6,242,259).Alternatively, the RNAs can be introduced as RNA or DNA molecules thatcan be expressed in the helper cell without integrating into the cell'sgenome. Methods of introduction include electroporation, viral vectors(e.g. SV40, adenovirus, nodavirus, astrovirus), and lipid-mediatedtransfection.

[0036] An alternative to multiple helper RNAs is the use of a singlenucleic acid molecule which encodes all the functions necessary forreplicating the viral replicon RNA and synthesizing the polypeptidesnecessary for packaging the alphaviral replicon RNA into infectivealphavirus replicon particles. This can be accomplished with an RNAmolecule determining the necessary functions or a DNA moleculedetermining the necessary functions. The single DNA helper nucleic acidcan be introduced into the packaging cell by any means known to the art,including but not limited to electroporation, lipid-mediatedtransfection, viral vectored (e.g. adenovirus or SV-40), and calciumphosphate-mediated transfection. Preferably, the DNA is introduced viathe electroporation-based methods of this invention, with voltage andcapacitance optimized for the cells and nucleic acid(s) beingintroduced. The DNA is typically electroporated into cells with adecrease in voltage and an increase in capacitance, as compared to thatrequired for the uptake of RNA. In all electroporations, the value forthe voltage and capacitance must be set so as to avoid destroying theability of the packaging cells to produce infective alphavirusparticles. The DNA was highly purified to remove toxic contaminants andconcentrated to about 5 mg/mL prior to electroporation. Generally, it ispreferable to concentrate the DNA to between 1-8 mg/mL, preferablybetween 5 and 8 mg/mL. The DNA helper is present in the electroporationmixture at from about 20-500, desirably from about 50 to about 300, forexample about 150 μg per 0.8 mL electroporation mixture, desirablycontaining from about 5×10⁷ to about 2×10⁸ cells, for example, about1.2×10⁸ cells.

[0037] Alternatively, the helper function, in this format and under aninducible promoter, can be incorporated into the packaging cell genomeprior to the introduction/expression of the viral RNA vector repliconnucleic acid, and then induced with the appropriate stimulus just priorto, concomitant with, or after the introduction of the RNA vectorreplicon.

[0038] Advantageously, the nucleic acid encoding the alphavirusstructural proteins, i.e., the capsid, E1 glycoprotein and E2glycoprotein, contains at least one attenuating mutation. The phrases“attenuating mutation” and “attenuating amino acid,” as used herein,mean a nucleotide mutation or an amino acid coded for in view of such amutation which result in a decreased probability of causing disease inits host (i.e., a loss of virulence), in accordance with standardterminology in the art, See, e.g., B. Davis, et al. Microbiology 132 (3ded. 1980), whether the mutation be a substitution mutation, or anin-frame deletion or addition mutation. The phrase “attenuatingmutation” excludes mutations which would be lethal to the virus unlesssuch a mutation is used in combination with a “restoring” mutation whichrenders the virus viable, albeit attenuated. In specific embodiments,the helper nucleic acid(s) include at least one attenuating mutation.

[0039] Methods for identifying suitable attenuating mutations in thealphavirus genome are known in the art. Olmsted et al. (1984; Science225:424) describes a method of identifying attenuating mutations inSindbis virus by selecting for rapid growth in cell culture. Johnstonand Smith (1988; Virology 162:437) describe the identification ofattenuating mutations in VEE by applying direct selective pressure foraccelerated penetration of BHK cells. Attenuating mutations inalphaviruses have been described in the art, e.g. White et al. 2001 J.Virology 75:3706; Kinney et al. 1989 Virology 70:19; Heise et al. 2000J. Virology 74:4207; Bernard et al 2000 Virology 276:93; Smith et al2001 J. Virology 75:11196; Heidner & Johnston 1994 J. Virology 68:8064;Klimstra et al. 1999 J. Virology 73:10387; Glasgow et al. 1991 Virology185:741; Polo and Johnston 1990 J. Virology 64:4438; and Smerdou andLiljestrom 1999 J. Virology 73:1092.

[0040] In certain embodiments, the replicon RNA comprises at least oneattenuating mutation. In other specific embodiments, the helper nucleicacid molecule(s) include at least one attenuating mutation. In theembodiment comprising two helper nucleic acid molecules, at least onemolecule includes at least one attenuating mutation, or both can encodeat least one attenuating mutation. Alternatively, the helper nucleicacid, or at least one of the first or second helper nucleic acidsincludes at least two, or multiple, attenuating mutations. Appropriateattenuating mutations depend upon the alphavirus used. For example, whenthe alphavirus is VEE, suitable attenuating mutations may be selectedfrom the group consisting of codons at E2 amino acid position 76 whichspecify an attenuating amino acid, preferably lysine, arginine, orhistidine as E2 amino acid 76; codons at E2 amino acid position 120which specify an attenuating amino acid, preferably lysine as E2 aminoacid 120; codons at E2 amino acid position 209 which specify anattenuating amino acid, preferably lysine, arginine, or histidine as E2amino acid 209; codons at E1 amino acid 272 which specify an attenuatingmutation, preferably threonine or serine as E1 amino acid 272; codons atE1 amino acid 81 which specify an attenuating mutation, preferablyisoleucine or leucine as E1 amino acid 81; and codons at E1 amino acid253 which specify an attenuating mutation, preferably serine orthreonine as E1 amino acid 253. Additional attenuating mutations includedeletions or substitution mutations in the cleavage domain between E3and E2 such that the E3/E2 polyprotein is not cleaved; this mutation incombination with the mutation at E1-253 is a preferred attenuated strainfor use in this invention. Similarly, mutations present in existing livevaccine strains, e.g. strain TC83 (see Kinney et al., 1989, Virology170: 19-30, particularly the mutation at nucleotide 3), are alsoadvantageously employed in the particles purified by the methods of thisinvention. An example of an attenuating mutation in the non-codingregion of the replicon nucleic acid is the substitution of A or C atnucleotide 3 in VEE.

[0041] Suitable helper and viral replicon RNAs are disclosed in U.S.Pat. No. 6,156,558, which is incorporated herein by reference.

[0042] Where the alphavirus is the South African Arbovirus No. 86 (S.A.AR86), suitable attenuating mutations may be selected from the groupconsisting of codons at nsP1 amino acid position 538 which specify anattenuating amino acid, preferably isoleucine as nsP1 amino acid 538;codons at E2 amino acid position 304 which specify an attenuating aminoacid, preferably threonine as E2 amino acid position 304; codons at E2amino acid position 314 which specify an attenuating amino acid,preferably lysine as E2 amino acid 314; codons at E2 amino acid position376 which specify an attenuating amino acid, preferably alanine as E2amino acid 376; codons at E2 amino acid position 372 which specify anattenuating amino acid, preferably leucine as E2 amino acid 372; codonsat nsP2 amino acid position 96 which specify an attenuating amino acid,preferably glycine as nsP2 amino acid 96; and codons at nsP2 amino acidposition 372 which specify an attenuating amino acid, preferably valineas nsP2 amino acid 372. Suitable attenuating mutations useful inembodiments wherein other alphaviruses are employed are known to thoseskilled in the art.

[0043] Attenuating mutations may be introduced into the nucleic acid byperforming site-directed mutagenesis, in accordance with knownprocedures. See, Kunkel, Proc. Natl. Acad. Sci. USA 82:488 (1985), thedisclosure of which is incorporated herein by reference in its entirety.Alternatively, mutations may be introduced into the nucleic acid byreplacement of homologous restriction fragments, in accordance withknown procedures, or by mutagenic polymerase chain reaction methods.

[0044] Once the helper nucleic acid(s) and replicon RNAs for use inproducing ARPs are generated, they are introduced into suitable hostcells, desirably by electroporation. The present inventors discoveredthat the electroporation carried out at relatively high cell densityallows efficient uptake of helper nucleic acid and virus replicon RNAs.The helper and replicon nucleic acids should be purified for use inelectroporation or other protocols for introducing the nucleic acidsinto cells for ARP production, but the helper RNAs need not be capped.

[0045] The step of producing the infectious viral particles in the cellsmay also be carried out using conventional techniques. See e.g., U.S.Pat. No. 5,185,440 to Davis et al., PCT Publication No. WO 92/10578 toBioption AB, and the U.S. Pat. No. 4,650,764 to Temin et al. (althoughTemin et al., relates to retroviruses rather than alphaviruses). Theinfectious viral particles may be produced by standard cell culturegrowth techniques improved by procedures described herein and/or byconventional particle harvesting techniques or the salt wash proceduredescribed hereinbelow. The salt wash appears to improve ARP recovery,especially when there are particular surface charges on the ARP surface.In the case of VEE, amino acid residues at E2=309 and E2-120 providegood sites for introducing a positive charge.

[0046] The viral replicon RNAs encode multiple heterologous codingsequences which are operably linked to promoters and other sequencesrequired for transcriptional and translational expression of the codingsequence in the host cell where the ARPS are to be introduced andexpressed.

[0047] Any amino acids which occur in the amino acid sequences referredto in the specification have their usual three- and one-letterabbreviations routinely used in the art: A, Ala, Alanine; C, Cys,Cysteine; D, Asp, Aspartic Acid; E, Glu, Glutamic Acid; F, Phe,Phenylalanine; G, Gly, Glycine; H, His, Histidine; I, lie, Isoleucine;K, Lys, Lysine; L, Leu, Leucine; M, Met, Methionine; N, Asn, Asparagine;P, Pro, Proline; Q, Gln, Glutamine; R, Arg, Arginine; S, Ser, Serine; T,Thr, Threonine; V, Val, Valine; W, Try, Tryptophan; Y, Tyr, Tyrosine.

[0048] As used herein “expression” directed by a particular sequence isthe transcription of an associated downstream sequence. If appropriateand desired for the associated sequence, there the term expression alsoencompasses translation (protein synthesis) of the transcribed RNA.Alternatively, different sequences can be used to direct transcriptionand translation.

[0049] Genomic DNA (where genes are not interrupted by introns and/orwhere this is not a significant proportion of the genome devoted tohighly repeated or non-expressed sequences) or cDNA is cloned into asuitably prepared virus vector nucleic acid preparation to produce arecombinant vector nucleic acid preparation. The recombinant vectornucleic acid preparation is then introduced into cells which allowpackaging of the recombinant vector nucleic acids into infectiveparticles. The recombinant vector nucleic acid preparation can beelectroporated into cells for packaging together with helper nucleicacids, RNA or DNA, in a relatively high cell density electroporation,e.g. about 10⁷ to about 10⁹ cells/per mL electroporation mixture. Thecells are then cultured in growth medium to allow packaging of therecombinant vector nucleic acids into viral replicon particles.

[0050] After the ARPs have been collected from the cells by salt wash,and desirably collected from the cell free supernatant, the ARPs arepartially purified by ion exchange chromatography.

[0051] The methods of the present invention are advantageously appliedto viral replicon nucleic acids derived from an alphavirus, preferablyfrom an attenuated alphavirus. A particularly preferred alphavirus isVenezuelan equine encephalitis virus (VEE). A specifically exemplifiedattenuated VEE is strain 3014, which virus or ARPs derived therefrom canbe purified using heparin affinity chromatography. VEE strain 3042 isanother attenuated virus suitable for use in ARP methods, but the coatof this virus or ARPs derived therefrom cannot be purified using heparinaffinity chromatography. The viruses, or ARPs derived therefrom, thatcarry mutations conferring glycosaminoglycan-binding ability areparticularly well suited for purification using the salt wash step, andthey can also be further purified using heparin affinity chromatography.

[0052] Cancers (neoplastic conditions) from which cells can be obtainedfor use in the methods of the present invention include carcinomas,sarcomas, leukemias, and cancers derived from cells of the nervoussystem. These include, but are not limited to: brain tumors, such asastrocytoma, oligodendroglioma, ependymoma, medulloblastomas, andPrimitive Neural Ectodermal Tumor (PNET); pancreatic tumors, such aspancreatic ductal adenocarcinomas; lung tumors, such as small and largecell adenocarcinomas, squamous cell carcinoma andbronchoalveolarcarcinoma; colon tumors, such as epithelialadenocarcinoma and liver metastases of these tumors; liver tumors, suchas hepatoma and cholangiocarcinoma; breast tumors, such as ductal andlobular adenocarcinoma; gynecologic tumors, such as squamous andadenocarcinoma of the uterine cervix, and uterine and ovarian epithelialadenocarcinoma; prostate tumors, such as prostatic adenocarcinoma;bladder tumors, such as transitional, squamous cell carcinoma; tumors ofthe reticuloendothelial system (RES), such as B and T cell lymphoma(nodular and diffuse), plasmacytoma and acute and chronic leukemia; skintumors, such as melanoma; and soft tissue tumors, such as soft tissuesarcoma and leiomyosarcoma.

[0053] The terms “neoplastic cell”, “tumor cell”, or “cancer cell”, usedeither in the singular or plural form, refer to cells that haveundergone a malignant transformation that makes them harmful to the hostorganism. Primary cancer cells (that is, cells obtained from near thesite of malignant transformation) can be readily distinguished fromnon-cancerous cells by well-established techniques, particularlyhistological examination. The definition of a cancer cell, as usedherein, includes not only a primary cancer cell, but also any cellderived from a cancer cell ancestor. This includes metastasized cancercells, and in vitro cultures and cell lines derived from cancer cells.When referring to a type of cancer that normally manifests as a solidtumor, a “clinically detectable” tumor is one that is detectable on thebasis of tumor mass; e.g., by such procedures as CAT scan, magneticresonance imaging (MRI), X-ray, ultrasound or palpation. Biochemical orimmunologic findings alone may be insufficient to meet this definition.

[0054] Pathogens to which multiple antigen immunological responses areadvantageous include viral, bacterial, fungal and protozoan pathogens.Viruses to which immunity is desirable include, but are not limited to,hemorrhagic fever viruses (such as Ebola virus), immune deficiencyviruses (such as feline or human immunodeficiency viruses),herpesviruses, coronaviruses, adenoviruses, poxviruses, retroviruses,aphthoviruses, nodaviruses, picornaviruses, orthomyxoviruses,paramyxoviruses, rubella, togaviruses, flaviviruses, bunyaviruses,reoviruses, oncogenic viruses such as retroviruses, pathogenicalphaviruses (such as Semliki forest virus or Sindbis virus),rhinoviruses, hepatitis viruses (Group B, C, etc), influenza viruses,among others. Bacterial pathogens to which immune responses are helpfulinclude, without limitation, staphylococci, streptococci, pneumococci,salmonellae, escherichiae, yersiniae, enterococci, clostridia,corynebacteria, hemophilus, neisseriae, bacteroides, francisella,pasteurellae, brucellae, mycobacteriae, bordetella, spirochetes,actinomycetes, chlamydiae, mycoplasmas, rickettsiae, and others.Pathogenic fungi of interest include but are not limited to Candida,cryptococci, blastomyces, histoplasma, coccidiodes, phycomycetes,trichodermas, aspergilli, pneumocystis, and others. Protozoans to whichimmunity is useful include, without limitation, toxoplasma, plasmodia,schistosomes, amoebae, giardia, babesia, leishmania, and others. Otherparasites include the roundworms, hookworms and tapeworms, filiaria andothers.

[0055] One of the strengths of the present alphavirus replicon vectortechnology is the ability to express more than one foreign gene. Untilnow, alphaviral replicon vaccines have been limited to the expression ofsingle or a handful of heterologous genes. This ability to express morethan one heterologous gene has been achieved through the addition ofmultiple promoter units to drive each individual gene's expression. Thenumber of heterologous genes a replicon vector can carry is ultimatelyconstrained by the capsid structure which is limited in the amount ofnucleic acid it can accommodate. An alternate strategy to singlereplicons expressing two or three antigens is to administer a cocktailof individual alphavirus replicon particles, each encoding andexpressing different antigens to elicit an immune response againstmultiple antigens and/or infectious agents as described herein. To date,these approaches have been limited to the expression of only a fewantigens at the same time (˜3), either in the multi-promoter or thecocktail replicon setting.

[0056] However, the recent improvements in process technology asdescribed herein for the generation of alphavirus replicon particleshave opened the door to new opportunities in vaccination againstmultiple antigens in the same vaccine preparation. The processimprovements are based on a high cell density electroporation method(cell concentration of 5×10⁷ to 1.5×10⁸ cells/mL of electroporationmixture) and salt wash techniques. Other improvements include the use ofuncapped (or capped) RNA molecules or DNA molecules in theelectroporation mixture. Yields from these improvements have beenincreased by 2 to 3 orders of magnitude (up to 10¹¹ i.u. can be producedfrom a single cuvette electroporation). These significant increases inefficiency of replicon production over the existing art mean a number ofvaccine approaches that were previously not feasible from a scalestandpoint are now enabled. The yield which can be achieved using thepresent methods, disclosed herein and in the referenced provisionalapplications, in theory, allows the production of ARPs which express thefull range of antigens expressed by the tumor, tumor cell, pathogen orparasite from which the nucleic acid inserted into the viral repliconnucleic acid was prepared.

[0057] One such approach is a “patient-specific vaccine” where a singlevaccine preparation is prepared on a patient-by-patient basis forprophylactic or therapeutic treatment of infectious diseases orneoplastic condition, e.g., cancer. Because a single tumor cell isestimated to express up to 5,000 genes, any attempt to generate analphaviral replicon tumor library vaccine expressing this large a numberof genes using traditional approaches would have been significantlylimited in the number of replicons expressing each gene. In addition,the particles would require purification to be suitable for formulationand administration in a clinical setting, and purification often resultsin a significant additional loss of titer. Using the improved ARPproduction techniques, we can now generate a population of repliconswhere most, if not all, genes from the tumor cell are likelyrepresented, on average, at least once in a population of 1×10⁵particles. In addition to the high yields from this approach, theprocess may provide a purer formulation on a per infectious unit basis.This means sequential purification steps may not be required, thuspreventing subsequent process losses. In addition, the increased puritymay lower the risk of eliciting anti-vector and anti-contaminant immuneresponses in the host. Normally, such a response could potentiallyprevent or compromise the efficacy of booster vaccinations. Forapproaches such as therapeutic tumor treatment, the ability to deliverhigh titers of vaccine in a pure formulation at frequent intervals is akey desirable characteristic of a vaccine. The present invention enablesa new multi-antigenic library approach to be taken using alphaviralreplicon vectors. These libraries can encode either multiple antigens,or entire gene repertoires from pathogenic organisms, parasites or tumorcells.

[0058] While prior art methods used to produce nucleic acids forintroduction into cells for ARP production are expensive and laborintensive, the present disclosure describes modifying various parametersto achieve improved ARP yield while simplifying the process anddecreasing the cost per ARP by orders of magnitude. The improvedalphavirus particle yield has enabled cloning nucleic acids derived froma tumor cell, pathogen or parasite into an alphavirus replicon nucleicacid and packaging with sufficient efficiency such that a representativeset of tumor cell, pathogen or parasite antigens are produced by the ARP“expression library”. The yield of ARPs is also sufficiently high suchthat a human or animal patient can be inoculated with an aliquot of suchan ARP preparation, with the preparation optionally further containingan immunological adjuvant, so that immune responses are generated to amultiplicity of antigenic determinants encoded within the ARP libraryand preparation administered to the patient.

[0059] Table 1 shows titration of multi-antigenic ARP produced from apool of cDNAs. Alphavirus replicon constructs expressing 10 differentheterologous genes (chloramphenicol acetyltransferase (CAT),beta-galactosidase (β-gal), Rat/Neu oncogene, luciferase, HIV Gag,cancer antigen A, and four malarial antigens: PkMSP1-42, PyHep17, PfAMA1and PkCSP) were linearized with Not1 restriction endonuclease, pooledand RNA transcripts generated using T7 RNA polymerase. The pool of RNAmolecules were co-electroporated into VERO cells with alphaviral capsidand glycoprotein helper RNAs to produce a population of ARP consistingof individual ARP expressing all 10 different antigens as determined byARP titration using immunofluorescence assays specific for each geneproduct.

[0060] Table 2 shows titration of multi-antigenic ARP produced from apool of RNAs. Alphavirus replicon constructs expressing 7 differentheterologous genes (CMV IE1, CMV gB, Influenza HA, HIV Pol, HIV Gag,Rat/neu, CAT) were individually linearized with Not1 restrictionendonuclease. RNA transcripts for each replicon were generated using T7RNA polymerase. The seven different RNA transcription products weremixed at equivalent concentrations and were co-electroporated into VEROcells with alphaviral capsid and glycoprotein helper RNAs. A populationof ARP was produced which expressed all 7 different antigens asdetermined by ARP titration using immunofluorescence assays specific foreach gene product.

[0061] Table 3 provides a summary of antigen-specific immune responsesin animals vaccinated with multi-antigenic ARP (as shown in FIG. 1).Antigen-specific immune responses in the form of humoral immunity aremeasured by either ELISA and presented as reciprocal geometric meantiter, or Western blot or IFA and presented as the lowest dilution atwhich antigen-specific signal was detectable. Antigen specific immuneresponses in the form of cellular immunity are measured by ELISPOTdetection of IFN-γ secreting cells and presented as antigen specificIFN-γ secreting lymphocytes per 10⁶ lymphocytes. Animals which receivedthe multi-antigenic ARP preparation either by a s.c. or an i.p. route ofinoculation mounted immune responses to all antigens in the preparation.As a positive control, one group received HIV-Gag ARP and mounted immuneresponses only specific for Gag. Negative control animals had nodetectable response to any antigen. Many samples were not titrated toendpoint, and are presented as titers equal to or greater than the givenvalue. Notably, the immune response elicited to the HIV Gag gene proteinas part of the multiantigenic preparations was equivalent on a humoraland cellular basis as compared to the HIV Gag protein delivered as asingle (homogeneous) standard preparation. This demonstrates codingsequences expressed as a component of a larger expression library canstill be effectively immunogenic employing the compositions and methodsof this invention.

[0062] The immunological ARP preparations which comprise expressiblenucleotide sequences encoding a multiplicity of tumor cell, pathogen orparasite antigenic determinants can be administered as a part of aprophylactic regimen, i.e., to lower the probability that the human oranimal to which the preparation is administered suffers from theneoplastic condition, pathogen infection or parasite infection, or as atherapeutic regimen, to lessen the severity of any conditions associatedwith an existing neoplastic condition, pathogen infection or parasiteinfection or such that the neoplastic condition, pathogen infection orparasite infection is prevented due to an immune response generated inthe human or animal to which the preparation has been administered.

[0063] While the generation of an immune response includes at least somelevel of protective immunity directed to the tumor cell (or neoplasticcondition), pathogen or parasite, the clinical outcome in the patientsuffering from such a neoplastic condition or infection with a parasiteor a pathogen can be improved by also treating the patient with asuitable chemotherapeutic agent, as known to the art. Where the pathogenis viral, an anti-viral compound such as acyclovir can be administeredconcomitantly with ARP vaccination, for example, in patients withherpesvirus infection, or HAART (highly active anti-retroviral therapy)in individuals infected with HIV. Where the pathogen is a bacterialpathogen, an antibiotic to which that bacterium is susceptible isdesirably administered and where the pathogen is a fungus a suitableantifungal antibiotic is desirably administered. Similarly, chemicalagents for the control and/or eradication of parasitic infections areknown and are advantageously administered to the human or animalpatients using dosages and schedules well known to the art. Where thepatient is suffering from a neoplastic condition, for example, a cancer,the administration of the immunogenic composition comprising ARPscapable of expressing a multiplicity of cancer-associated antigens inthe patient to which it has been administered is desirably accompaniedby administration of antineoplastic agent(s), including, but not limitedto, such chemotherapeutic agents as daunorubicin, taxol, thioureas,cancer-specific antibodies linked with therapeutic radionuclides, withthe proviso that the agent(s) do not ablate the ability of the patientto generate an immune response to the administered ARPs and the antigenswhose expression they direct in the patient.

[0064] Pharmaceutical formulations, such as vaccines or otherimmunogenic compositions, of the present invention comprise animmunogenic amount of the infectious, propagation-defective alphavirusreplicon particles in combination with a pharmaceutically acceptablecarrier. An “immunogenic amount” is an amount of the infectiousalphavirus particles which is sufficient to evoke an immune response inthe subject to which the pharmaceutical formulation is administered. Anamount of from about 10¹ to about 10¹⁰ infectious units per dose,preferably 10⁵ to 10⁸, is believed suitable, depending upon the age andspecies of the subject being treated. Exemplary pharmaceuticallyacceptable carries include, but are not limited to, sterile pyrogen-freewater and sterile pyrogen-free physiological saline solution. Subjectswhich may be administered immunogenic amounts of the infectious,propagation defective alphavirus particles of the present inventioninclude but are not limited to human and animal (e.g., dog, cat, horse,pig, cow, goat, rabbit, donkey, mouse, hamster, monkey) subjects.Immunologically active compounds such as cytokines and/or BCG can alsobe added to increase the immune response to the administered viralreplicon particle preparation. Administration may be by any suitablemeans, such as intratumoral, intraperitoneal, intramuscular,intradermal, intranasal, intravaginal, intrarectal, subcutaneous orintravenous administration.

[0065] Immunogenic compositions comprising the ARPs (which direct theexpression of the antigens of interest when the compositions areadministered to a human or animal) produced using the methods of thepresent invention may be formulated by any of the means known in theart. Such compositions, especially vaccines, are typically prepared asinjectables, either as liquid solutions or suspensions. Solid forms, forexample, lyophilized preparations, suitable for solution in, orsuspension in, liquid prior to injection may also be prepared.

[0066] The active immunogenic ingredients (the ARPs) are often mixedwith excipients or carriers that are pharmaceutically acceptable andcompatible with the active ingredient. Suitable excipients include butare not limited to sterile water, saline, dextrose, glycerol, ethanol,or the like and combinations thereof.

[0067] In addition, if desired, the vaccines may contain minor amountsof auxiliary substances such as wetting or emulsifying agents, pHbuffering agents, and/or adjuvants which enhance the effectiveness ofthe vaccine. Examples of adjuvants which may be effective include butare not limited to: aluminum hydroxide;N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP);N-acetyl-nor-muramyl-L-alanyl-D-isoglutamine (CGP 11637, referred to asnor-MDP);N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3hydroxyphosphoryloxy)-ethylamine(CGP 19835A, referred to as MTP-PE); and RIBI, which contains threecomponents extracted from bacteria, monophosphoryl lipid A, trehalosedimycolate and cell wall skeleton (MPL+TDM+CWS) in a 2% squalene/Tween80 emulsion. The effectiveness of an adjuvant may be determined bymeasuring the amount of antibodies directed against the immunogenicproduct of the ARP resulting from administration of the immunogen invaccines which are also comprised of the various adjuvants. Suchadditional formulations and modes of administration as are known in theart may also be used.

[0068] One or more immuno-potentiator molecules, such as chemokinesand/or cytokines, can be incorporated into the immunogenic compositionadministered to the patient or animal. Alternatively, alphavirusreplicon vectors which contain coding sequence(s) for theimmuno-potentiator molecule can be incorporated in the immunogeniccomposition. It is understood that the choice of chemokine and/orcytokine may vary according to the neoplastic tissue or cell, parasiteor pathogen against which an immune response is desired. Examples caninclude, but are not limited to, interleukin-4, interleukin-12,gamma-interferon, granulocyte macrophage colony stimulating factor andFLT-3 ligand.

[0069] The immunogenic (or otherwise biologically active) ARP-containingcompositions are administered in a manner compatible with the dosageformulation, and in such amount as will be prophylactically and/ortherapeutically effective. The quantity to be administered, which isgenerally in the range of about 10¹ to about 10¹⁰ infectious units,preferably 10⁵ to 10⁸, in a dose, depends on the subject to be treated,the capacity of the individual's immune system to synthesize antibodies,and the degree of protection desired. Precise amounts of the activeingredient required to be administered may depend on the judgment of thephysician, veterinarian or other health practitioner and may be peculiarto each individual, but such a determination is within the skill of sucha practitioner.

[0070] The vaccine or other immunogenic composition may be given in asingle dose or multiple dose schedule. A multiple dose schedule is onein which a primary course of vaccination may include 1 to 10 or moreseparate doses, followed by other doses administered at subsequent timeintervals as required to maintain and or reinforce the immune response,e.g., at weekly, monthly or 1 to 4 months for a second dose, and ifneeded, a subsequent dose(s) after several months or years.

[0071] Standard techniques for cloning, DNA isolation, amplification andpurification, for enzymatic reactions involving DNA ligase, DNApolymerase, restriction endonucleases and the like, and variousseparation techniques are those known and commonly employed by thoseskilled in the art. A number of standard techniques are described inSambrook et al. (1989) Molecular Cloning, Second Edition, Cold SpringHarbor Laboratory, Plainview, N.Y.; Maniatis et al. (1982) MolecularCloning, Cold Spring Harbor Laboratory, Plainview, New York; Wu (ed.)(1993) Meth. Enzymol. 218, Part I; Wu (ed.) (1979) Meth. Enzymol. 68; Wuet al. (eds.) (1983) Meth. Enzymol. 100 and 101; Grossman and Moldave(eds.) Meth. Enzymol. 65; Miller (ed.) (1972) Experiments in MolecularGenetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York;Old and Primrose (1981) Principles of Gene Manipulation, University ofCalifornia Press, Berkeley; Schleif and Wensink (1982) Practical Methodsin Molecular Biology; Glover (ed.) (1985) DNA Cloning Vol. I and II, IRLPress, Oxford, UK; Hames and Higgins (eds.) (1985) Nucleic AcidHybridization, IRL Press, Oxford, UK; Setlow and Hollaender (1979)Genetic Engineering: Principles and Methods, Vols. 1-4, Plenum Press,New York; and Ausubel et al. (1992) Current Protocols in MolecularBiology, Greene/Wiley, New York, N.Y. Abbreviations and nomenclature,where employed, are deemed standard in the field and commonly used inprofessional journals such as those cited herein.

[0072] All references cited in the present application are incorporatedby reference in their entireties to the extent that they are notinconsistent with the present disclosure.

[0073] The following examples are provided for illustrative purposes,and are not intended to limit the scope of the invention as claimedherein. Any variations in the exemplified articles which occur to theskilled artisan are intended to fall within the scope of the presentinvention.

EXAMPLES Example 1 Generation of Alphavirus Replicon Vectors Expressinga Library of Tumor Associated Antigens

[0074] Tumor cells are typically obtained from a cancer patient byresection, biopsy, or endoscopic sampling; the cells may be useddirectly, stored frozen, or maintained or expanded in culture RNA isextracted from tumor cells using standard methods known in the art, e.g.using commercially available reagents and kits such as Trizol (Sigma,St. Louis, Mo.) or S.N.A.P. total RNA isolation kit (Invitrogen, Inc,Carlsbad, Calif.), followed by mRNA purification on oligo(dT)-Sepharose. mRNA can be further enriched in tumor-specific sequencesby subtractive hybridization or other method known in the art.First-strand cDNA is synthesized using oligo (dT) oligonucleotides witha rare restriction site at 5′-terminus. Following purification of thecDNA, the second strand is produced using any of the standard methods,e.g. using DNA polymerase I-RnaseH or non-specific amplification. Anadaptor is then ligated to create a cohesive end, and double-strandedDNA is digested with a rarely recognized restriction endonuclease (suchas DraI) at a site which has been incorporated in the oligo (dT) primer.This procedure creates a double-stranded cDNA with non-compatiblecohesive ends suitable for directional cloning.

[0075] Alternatively, a strategy described in Example 2 (below) can beused for generation of cohesive ends for directional cloning. In anadditional embodiment, cohesive ends can be attached by terminaldeoxyribonucleotide transferase. The double-stranded cDNA is then clonedinto a plasmid replicon vector or used to construct recombinant repliconmolecules in vitro in a manner similar to the one described below. Thisapproach produces recombinant replicon molecules that contain a biotinlabel on the 3′-termini and a T7 promoter on the 5′-termini, thusallowing for selection of the recombinant molecules and generation ofRNA in vitro using T7 DNA-dependent RNA polymerase. Additional selectivesteps can be implemented to “down-select” the number of antigens presentin the tumor antigen library. Methods such as subtractive hybridizationand differential analysis are well known in the art (See U.S. Pat. Nos.5,958,738, 5,827,658 and 5,726,022 and U.S. patent app. 2002-0018766),and such a selection method can be implemented immediately prior tocloning into the VEE replicon construct. This approach serves to limitthe tumor antigen pool to genes either exclusively expressed orpreferentially up-regulated in a tumor cell. This selection serves toreduce or eliminate the frequency and/or presence of normal cellulargenes in the antigen library. Without wishing to be bound by anyparticular theory, it is believed that additional benefits include theelimination of non-tumor specific antigens focusing of the immuneresponse against tumor-associated antigens, thus maximizing thepotential specificity of the vaccine preparation and reducing the riskof inducing autoimmune responses. This “down-selection” of the antigenrepertoire is also relevant to prime-boost strategies. In manyinstances, it may be advantageous to vaccinate with a broad array oftumor antigens, and in the subsequent boost inoculations, tolimit/down-select the number of antigens so as to effectively focus theimmune system on specific antigens. This can feasibly be done bydown-selecting antigens also based on identifying which antigens thehost has responded to following the first immunization, and thusessentially tailoring each subsequent boost to augment the immuneresponse to antigens the host has demonstrated it can recognize and towhich an immune response has been raised.

Example 2 Generation of Alphavirus Replicon Vectors Expressing cDNAsSpecific for Infectious Disease Organism from a Sample of InfectedTissue or Blood When the Target Gene Sequences Are Known

[0076] This example describes cloning of a viral/bacterial/parasiticgene repertoire specific for an individual with either an acute orchronic infection in instances where the gene or genes of interest(i.e., the genes which encode the immunogenic moieties to be expressedby the replicons) are acquired from an agent of known sequence. An mRNAis isolated from a tissue or blood sample following standard methodsknown in the art, e.g. S.N.A.P. total RNA isolation kit (Invitrogen,Inc, Carlsbad, Calif.). First-strand cDNA is synthesized by any standardmethods known in the art, e.g. cDNA cycle kit (Invitrogen, Inc,Carlsbad, Calif.), or using AMV reverse transcriptase and randomprimers. The gene(s) of interest are amplified from cDNA using targetgene-specific primers, following which the amplicon is purified using aPCR purification kit (Qiagen Inc., Valencia, Calif.) or any other methodknown in the art. This amplicon can be cloned into the VEE repliconusing methods known to those skilled in the art, e.g. using G:C cloning,directional cloning following restriction endonuclease digestion or invitro recombination methods such as Gateway (Invitrogen, Carlsbad,Calif.) or the Cre-lox recombination system.

[0077] In a preferred embodiment, the coding sequence(s) of interest areamplified using RNA/DNA hybrid oligonucleotides. Followingamplification, the DNA amplicon is treated with NaOH to digest the RNAportion of the primers, or alternatively, incubated at 50° C. in thepresence of rare-earth metals to selectively hydrolyze thephosphodiester bond between the deoxyribonucleotide and theribonucleotide (Chen et al., 2000, Biotechniques; 28(3):498-500, 504-5and Chen et al., 2002, Biotechniques, 32:516, 518-20) in order to createa 3′-overhang required for ligation. A complementary 3′-overhang in thevector sequences is created in a similar fashion or by using arestriction endonuclease. In this manner the two fragments of thereplicon molecule are prepared: the left arm and the right arm. The leftarm includes a T7 promoter operatively linked to VEE specific sequences,up to and including a convenient cloning site. The right arm containsthe 3′-untranslated region of VEE. The right arm also contains a biotinlabel at the 3′-terminus. The amplified fragment with a 3′-overhang islinked to the left and right arms of the vector using T4 DNA ligase. Theassembled molecule is separated from the ligation reaction mixture usingmagnetic streptavidin-coated beads, or any other similar solid-phaseabsorption technique. Full-length replicon RNA is produced from purifiedrecombinant vector DNA by in vitro transcription using T7 DNA-dependentRNA polymerase. This step results in production of only full-lengthrecombinant molecules, since incomplete molecules do not bind tostreptavidin, or are not transcribed due to the lack of T7 promotersequences. The resulting recombinant replicon RNA molecules encode acomprehensive repertoire of the target gene(s), which represent thegenotype of the target which is infectious in the patient. An advantageof this method is the ability to have representation of all variants fora particular gene population from an individual, e.g. amplification ofthe HIV-1 envelope gp160 gene sequence isolated from an HIV-infectedpatient using the methods outlined above generates an ARP populationencoding the majority or all of the envelope variants from thatparticular patient. If the patient is infected with multiple strains ofvirus or distinct variants originating from an original parentalcirculating strain, the technique above captures all variants and theyare represented in the final ARP vaccine population.

Example 3 Generation of Alphavirus Replicon Vectors ExpressingInfectious Disease Specific cDNA From a Sample of Infected Tissue/BloodWhen the Target Gene Sequences Are Not Known

[0078] This example describes cloning of a viral/bacterial/parasiticgene repertoire in cases where the gene or genes of interest are not ofa known sequence. Viral, bacterial or parasitic mRNA is isolated from afield sample or a stock culture or purified preparation usingMICROBExpress kit (Ambion, Austin, Tex.) or any other method known tothose skilled in the art. First strand cDNA is synthesized using randomprimers, or random primers with a rare restriction site at the5′-terminus, followed by second-strand cDNA synthesis with DNApolymerase I and RNase H using standard methods known to one skilled inthe art. Double-stranded cDNA is subsequently cloned into a VEE vectorafter ligation of an adaptor or a linker sequence as follows. In caseswhen the cDNA is synthesized with a random primer containing a rarerestriction site, a linker is used to attach a second different rarerestriction site at the 5′-terminus of double-stranded cDNA. Digestionof the cDNA pool with these two restriction endonucleases results in thegeneration of cDNA fragments with different cohesive ends, whichfacilitates directional cloning into the replicon vector using methodsknown in the art. In the case that cDNA is generated with a randomprimer lacking an additional unique restriction site, double-strandedcDNA is methylated using EcoRI methylase to protect internal sequencesfrom subsequent digestion with EcoRI restriction enzyme. The EcoRIlinker is then attached using T4 DNA ligase, followed by digestion withEcoRI restriction endonuclease. This produces a cDNA fragment withcohesive ends, which can be cloned into a replicon. A cloning strategysimilar to the one described in Example 2 can be used for the generationof a pool of replicon molecules labeled with biotin at the 3′-terminusand containing a T7 DNA-dependent RNA polymerase promoter at the5′-terminus. Again, as described in the previous examples, subtractivehybridization or differential display can be used as additionalsubsequent screening steps to positively or negatively select pathogenspecific genes/sequences in a manner similar to that described for thetumor specific approaches. Again, this can be done with all vaccinationsor on a “real-time” basis where the host is monitored duringvaccinations and the vaccine is tailored to contain antigens to whichthe host demonstrates recognition and response.

Example 4 Multi-Antigenic ARP Packaging

[0079] Generation of a population of ARPs in which each ARP expresses adifferent antigen or antigens from a single electroporation event wereperformed in two alternate manners. The first method consisted ofcombining 0.5 μg of DNA from 10 different replicon vector constructs,each containing a single heterologous coding sequence (Table 1). TheDNAs were linearized with NotI restriction enzyme, and RNA wastranscribed from the replicon DNA pool with T7 RNA polymerase. Themultiple-replicon RNA transcription reaction was then purified using anRNEasy column (Qiagen Inc., Valencia, Calif.). ARP were produced byelectroporation using 30 μg of multiple-replicon RNA combined with 30 μgeach of purified capsid (C) helper and glycoprotein (GP) helper RNAsinto 1.0×10⁸ Vero cells in a 0.8 mL volume cuvette. Afterelectroporation, the cells were suspended in 200 mL of Opti-pro media(Invitrogen, Carlsbad, Calif.) and seeded into 4, 175 cm² cultureflasks. Approximately 26 hr post electroporation the media from eachflask was discarded and replaced with 5 mL of a salt wash solution (1 MNaCl in 20 mM phosphate buffer (pH 7.3). The flasks were incubated atroom temperature for 10 minutes, the salt wash was collected andfiltered through a 0.2 micron syringe filter. The titer of individualARP was determined in Vero cells using antigen-specific antibodies bystandard immunofluorescence methods. The titer of each ARP in the poolproduced from a single electroporation is shown in Table 1. The titer ofthe ARP preparation was 4.1×10⁹ infectious units per mL, resulting in atotal of 8.2×10¹⁰ i.u. total ARP generated from a single cuvetteelectroporation. Representatives of all 10 antigens were present in theARP population. This example demonstrates that not only can multipledifferent antigens be expressed from a single ARP preparation, but thatthe range of antigen type can be extremely varied. In this preparationantigens were derived from viral infectious disease origin (HIV), fromparasitic origin (malaria), or from cancer origins (rat/Neu and cancerantigen A) as well as enzymes (CAT, luciferase and β-gal).

[0080] The second method consisted of generating RNA transcripts foreach replicon vector independently rather than as a pool. The 7 repliconvectors used in this experiment are listed in Table 2. 10 μg of eachpurified replicon RNA was combined with 30 μg each of purified C-helperand GP-helper RNAs for a total of 130 μg of RNA. The RNA mix was thenelectroporated into 1.0×10⁸ Vero cells. Electroporated cells weresuspended in 200 mL of Opti-pro media and seeded into 2 300 cm² cultureflasks. Approximately 24 hr post electroporation the media from eachflask was collected and replaced with 10 mL of salt wash (1M NaCl in 20mM phosphate buffer, pH 7.3). The flasks were incubated at roomtemperature for 5 minutes, and the salt wash was collected. Both themedia and salt wash material were filtered through a 0.2 micron syringefilter. The individual ARP in both the media and salt wash were titratedin Vero cells using antigen specific antibodies for IFA. The titer ofeach ARP found in either the media or salt wash is shown in Table 2. Thetiter of the ARP recovered in the media was 5.3×10⁷ i.u./mL resulting in1.1×10¹⁰ i.u. total ARP generated (5.3×10⁷ i.u./mL×200 mL=1.1×10¹⁰i.u.). The titer of the ARP recovered in the salt wash was 4.05×10⁹i.u./mL resulting in 8.1×10¹⁰ i.u. total ARP generated per singlecuvette electroporation. The material in the salt wash and the mediawere combined for a total of 9.2×10¹⁰ i.u. ARP. Representatives of all 7antigens were present in the ARP population. The pooled ARP were thenpurified on a HiTrap Heparin HP 5 mL column (Amersham Bioscience,Uppsala, Sweden) for use in animal vaccination studies.

[0081] ARP preparations were all evaluated by standard safety testing toconfirm the absence of replication competent virus (RCV). Briefly, 1×10⁸i.u. of each preparation was inoculated onto VERO cell monolayers at anm.o.i. of less than 1 for 1 hour. Growth media was applied to the cellmonolayers after a 1 hour infection period and cells cultured for 24hours. After 24 hours, the entire supernatant was harvested, clarifiedand applied to fresh VERO cell monolayers for a further 48 hours. Cellmonolayers were monitored for the presence of any cytopathic effect(CPE) indicative of the presence of contaminating replication competentvirus particles. In all cases, no RCV was detected in anymulti-antigenic ARP vaccine preparations.

Example 5 Animal Studies With Multi-Antigenic Virus Particles

[0082] Five to six week-old female BALB/c mice were obtained fromCharles River Laboratories and were acclimatized for one week prior toany procedure. Mice were fed ad libitum water (reverse osmosis, 1 ppmCl) and an irradiated standard rodent diet (NIH31 Modified andIrradiated) consisting of 18% protein, 5% fat, and 5% fiber. Mice werehoused in static microisolators on a 12-hour light cycle at 21-22° C.(70-72° F.) and 40%-60% humidity. All animal studies comply withrecommendations of the Guide for Care and Use of Laboratory Animals withrespect to restraint, husbandry, surgical procedures, feed and fluidregulation, and veterinary care. The animal care and use program isAAALAC accredited.

[0083] For prime and boost injections, groups of mice were eachinoculated in both rear footpads under isoflorane anesthesia withmulti-antigenic ARP in diluent (PBS with 1% v/v human serum albumin and5% w/v sucrose). Footpad subcutaneous (s.c.) injections were performedwith a 30.5 G needle and a 0.10 mL Hamilton syringe by injecting 20 μLin each footpad. Intraperitoneal (i.p.) inoculations were administeredby the same syringe/needle but in a volume of 0.1 mL. Animals wereinoculated on days 1, 23 and 44. Serum samples were obtained byretro-orbital bleeding under isoflorane anesthesia before the firstinoculation on days −7 and 0 (pre-bleed), days 30 and 35 (after theprimary inoculation) and days 51 and 56 (7 and 12 days after the boost).Spleens were harvested at least 7 days post-boost for IFN-γ ELISPOTassays.

[0084] Immunofluorescence assay (IFA) of ARP-infected Vero cells wasused to measure the potency or infectious titer of each of the vaccinepreparation. All ARP vaccines were titered prior to inoculations. On theday of each injection residual inocula were back-titrated. ARP vaccineinocula were kept at 4° C. during the time following vaccination tomaintain titer. Test groups included the following vaccine preparations:high and low dose multi-antigenic ARP preparations administered asdosages of 1×10⁸ or 1×10⁶ i.u., respectively. As a control formonitoring the immune response as compared to a single ARP preparationexpressing a single antigen ARP expressing HIV Gag alone wereadministered at a dosage equivalent to the number of HIV-Gag ARP in themulti-antigenic mix. Negative control animals were sham immunized withdiluent alone.

Example 6 Measurement of Humoral and Cellular Immune Responses afterMulti-Antigenic ARP Administration

[0085] Detection of HIV Gag specific antibodies by ELISA. Purifiedrecombinant histidine-tagged (his)-p55 from HIV-1 subtype C isolateDU-422 was used as antigen coat. Briefly, BHK cells were transfectedwith VEE replicon RNA expressing his-p55 and Triton-X 100 lysates wereprepared. Protein was purified by ion metal affinity chromatography, inaccordance with the suppliers′ recommendations.

[0086] Sera from Day 51 (7 days post boost) were evaluated for thepresence of Gag-specific antibodies by a standard indirect ELISA. Fordetection of Gag-specific total Ig, a secondary polyclonal antibody thatdetects IgM, IgG and IgA was used for end point titer determination.Briefly, 96-well Maxisorp ELISA plates (polystyrene multiwell plateswith modified surface to increase affinity for polar molecules, i.e.,antibodies; Nunc, Naperville, Ill.) were coated with 50 μL of 0.05 Msodium carbonate buffer, pH 9.6 (Sigma, St. Louis, Mo.) containing 40-80ng his-p55 per well. Plates were covered with adhesive plastic andincubated overnight at 4° C. The next day, unbound antigen was discardedand plates were incubated for 1 hour with 200 μL blocking buffer (PBScontaining 3% w/v BSA) at room temperature. Wells were washed 6 timeswith PBS and 50 μL/well of test serum, diluted serially two-fold inbuffer (PBS with 1% w/v BSA and 0.05% v/v Tween 20), was added toantigen-coated wells. Mouse anti-p24 monoclonal antibody (Zeptometrix,Buffalo, N.Y.) was included in every assay as a positive control.Negative controls in each assay included blanks (wells with all reagentsand treatments except serum) and pre-bleed sera. Plates were incubatedfor one hour at room temperature, and then rinsed 6 times with PBS. 50μL /well of alkaline phosphatase (AP)-conjugated goat anti-mousepoly-isotype secondary antibody (Sigma) diluted to a predeterminedconcentration in diluent buffer was added to each well and incubated for1 hour at room temperature. Wells were rinsed 6 times with PBS beforeaddition of 100 μL p-nitrophenyl phosphate (pNPP) substrate (Sigma). Theserum antibody ELISA titer was defined as the inverse of the greatestserum dilution giving an optical density at 405 nm greater than or equalto 0.2 above the background (blank wells). Positive antibody (immune)responses were detected in mice vaccinated with the multi-antigenic ARPpreparation and in mice that received the ARP HIV-Gag.

[0087] Gag and Pol antigen-specific Interferon-gamma (IFN-γ) secretingcells are detected by IFN-γELISPOT Assay. Single-cell suspensions ofsplenic lymphocytes from ARP-immunized BALB/c mice were prepared byphysical disruption of the splenic capsule in R-10 medium (RPMI medium1640 supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, 0.1mM MEM non-essential amino acids solution, 0.01 M HEPES, 2 mM glutamineand 10% heat inactivated fetal calf serum). Lymphocytes were isolated byLympholyte M density gradient centrifugation (Accurate Scientific,Westbury, N.Y.), washed twice and resuspended in fresh R-10 medium.Total, unseparated splenic lymphocyte populations were tested.

[0088] A mouse IFN-γ ELISPOT kit (Monoclonal Antibody Technology, Nacka,Sweden) was used to perform the assay. Viable cells were seeded intoindividual ELISPOT wells in a Multiscreen Immobilon-P ELISPOT plate(ELISPOT certified 96-well filtration plate with high protein-bindingPVDF membranes; Millipore, Billerica, Mass.) that had been pre-coatedwith an anti-IFN-γ monoclonal antibody, and incubated for 16-20 hours.Cells were removed by multiple washes with buffer and the wells wereincubated with a biotinylated anti-IFN-γ monoclonal antibody, followedby washing and incubation with Avidin-Peroxidase-Complex (Vectastain ABCPeroxidase Kit, Vector Laboratories, Burlingame, Calif.). Followingincubation, the wells were washed and incubated for 4 minutes at roomtemperature with substrate (Avidin-Peroxidase Complex tablets, Sigma,St. Louis, Mo.) to facilitate formation of spots, which represent thepositions of the individual IFN-γ-secreting cells during culture. Plateswere enumerated by automated analysis with a Zeiss KS ELISPOT system.

[0089] To enumerate Gag-specific IFN-γ secreting cells in lymphocytesfrom mice immunized with HIV GAG ARP and multi-antigenic HIV ARPconstructs expressing gag, lymphocytes were stimulated with theimmunodominant CD8 H-2K^(d)-restricted HIV-Gag peptide, or an irrelevantNef peptide pool (Nef peptide containing 10 15-mers overlapping by 11made from Clade C HIV strain DU₁₅₁), for 16-20 hours (5% CO₂ at 37° C).The Gag peptide was tested at 10 μg/mL and the Nef control was tested at20 μg/mL. Cells minus peptide serve as a background control. As apositive control, cells were stimulated with 4 μg/mL concanavalin A fora similar time period. Peptides were synthesized and purified to >90%(New England Peptide, Gardner, Mass.).

[0090] To enumerate Pol-specific IFN-γ secreting cells in lymphocytesfrom mice immunized with multi-antigenic ARP constructs expressing pol,the protocol above was used with the following modifications. HIV-1 Polepitopes for both CD8 and CD4 T cells have been recently identified inthe H-2^(d) background (Casimiro et al., J. Virology 76:185, 2002). Cellpopulations were stimulated with a pool of 3 Pol epitope-containingpeptides and with an irrelevant antigen peptide pool as a negativecontrol (nef pool 1). The three peptides below were selected after aliterature search to identify the known murine Pol CTL epitopes.VYYDPSKDLIA (SEQ ID NO:1) (Casimiro et al, J. Virol. 76:185, 2002)ELRQHLLRWGL (SEQ ID NO:2) (Casimiro et al, J. Virol. 76:185, 2002)ELREHLLKWGF (SEQ ID NO:3) (homologue to number 2, identical to oursequence).

[0091] These three peptides were mixed together at a concentration of 10μg/mL each (total peptide concentration was 30 μg/mL) and added totriplicate wells. The ELISPOT assay results presented were performed 26days post the second boost.

[0092] Detection of Rat/neu specific antibodies was by ELISA. Rat/neuantigen for use as an ELISA reagent was prepared as follows: a histidinetag was added by PCR to the C-terminus of the Rat/neu coding sequence inpRAT/neu #14. This PCR amplified product was digested and ligated intothe VEE replicon plasmid, pERK. BHK cells were electroporated with RNAgenerated from the pERK Rat/neu-his construct. At 16 hourspost-electroporation cell lysates were prepared and purified over anickel affinity column, achieving 60-70% purity of the his-tagged Ratneu antigen.

[0093] Sera from Day 51 (7 days post boost) were evaluated for thepresence of Rat/neu-specific antibodies by an indirect ELISA. Nunc highbinding plates were coated at 4° C. overnight with 75 ng/well ofhis-tagged Rat neu in carbonate-bicarbonate coating buffer. The next dayplates were blocked with 200 μl/well of 3% BSA in PBS for 1 hour at 30°C. After 6 washes in PBS, 50 μl of mouse serum samples were diluted in1% BSA, 0.05% Tween 20 in PBS and added to each well and the plates wereincubated for 1 hr at 30° C. Pre-bleeds at 1:40 and 1:80, as well astwo-fold dilutions from 1:40-1:1280 of day 51 sera were tested for eachexperimental animal. Plates were then washed 6 times with PBS, followedby the addition of 50 μl/well of a 1:500 dilution of goat anti-mouse HRPand incubated for 1 hr at 30° C. Plates were washed as before anddeveloped with 100 μl/well of ABTS (KPL), and the absorbance was read at405 nm using a standard ELISA reader. The cut off value to determine apositive sample was determined by averaging the OD (absorbance) value ofall the pre-bleed serum samples diluted 1:40 and multiplying that valueby two. Any sample with an OD greater than the cut off value wasconsidered positive.

[0094] Detection of anti-CAT specific antibodies was by ELISA. Ananti-CAT antibody ELISA was developed to detect anti-CAT immuneresponses in multi-antigen ARP vaccinated mice. ELISA microplates coatedwith sheep anti-CAT polyclonal antibodies (Roche, Indianapolis, Ind.)were loaded with 0.15 ng of purified CAT protein suspended in CAT ELISAsample buffer (Roche) in a volume of 50 μl per well. The ELISA plateswere incubated at 37° C. for 45 min and washed three times with 0.2 mLof CAT ELISA wash buffer (Roche). 50 μl of mouse serum, two-foldserially diluted in sample buffer, was loaded per well and the plateswere incubated at 37° C. for 45 min. After incubation, the ELISA plateswere washed three times as described above. Goat anti-mouseHRP-conjugated secondary antibody (Kirkegaard and Perry Laboratories(KPL), Gaithersburg, Md.) diluted 1:500 in sample buffer was added toeach well (0.1 mL per well) and incubated at 37° C. for 45 min. Afterincubation, the plates were washed three times as described above, and0.1 mL of ABTS peroxidase substrate (2,2′-azino-bis3-ethylbenzthiazoline-b-sulfonic acid; KPL) was added per well. Colordevelopment was ended by addition of 0.1 mL stop solution (KPL) and theabsorbance in the plates were read at 405 nm using a Molecular DevicesVersamax microplate reader. The cut off value to determine a positivesample was determined by averaging the OD value of all the pre-bleedserum samples diluted 1:40 and multiplying that value by two. Any samplewith an OD greater than the cut off value was considered positive.

[0095] Detection of CMV gB specific antibodies was by Western blot.Analysis of anti-gB immune responses in multi-antigen ARP vaccinatedanimals was by Western blot. Purified, recombinant, histidine-tagged gBprotein was electrophoresed through 4-10% Bis-Tris NuPAGE gels (Sodiumdodecyl sulfate-polyacrylamide gel; Invitrogen, Carlsbad, Calif.) andtransferred to PVDF membranes using a Novex mini-cell (Invitrogen)electrophoresis unit. Pre-bleed and Day 51 post-vaccination sera werediluted 1:40 or 1:80 for each animal in blocking buffer (Invitrogen) andincubated on strips of PVDF membranes after gB protein transfer. Goatanti-mouse alkaline phosphates conjugated antibody (Sigma, St. Louis,Mo.) diluted 1:10,000 in blocking buffer was used as the secondaryantibody. Western blots were developed using BCIP/NBT(5-bromo,4-chloro,3-indolylphosphate/nitroblue tetrazolium; Bio Rad,Hercules, Calif.), and color development was arrested by washing withdistilled water. Positive samples were identified by visual detection ofimmunoreactive bands with electrophoretic mobility matching the expectedmolecular weight of gB on the immunoblot.

[0096] Detection of Influenza HA specific antibodies was byimmunofluorescence assay (IFA). Analysis of anti-HA immune responses inmulti-antigen ARP vaccinated animals was determined by IFA. Vero cellswere electroporated with a VEE replicon vector that expressed the H1N1influenza HA gene and 1×10⁴ electroporated cells per well were seededinto 96 well tissue culture plates. Electroporated Vero cells were fixedwith methanol 16 hr post-electroporation. Pre-bleed and day 56post-vaccination sera were diluted two-fold from 1:40 to 1:160 inblocking buffer (PBS:FBS (1:1)) for each animal and incubated on HAprotein expressing Vero cells. A goat anti-mouse Alexa Fluor 488conjugated antibody (Molecular Probes, Eugene, Oreg.) diluted 1:400 wasused as the secondary antibody. Cells were analyzed on a Nikon EclipseTE300 UV microscope for HA specific fluorescence. Titer was determinedby visual detection of immunofluorescent cells at the lowest detectableserum dilution value.

[0097] Detection of anti-CMV IE1 specific antibodies was by ELISA.Purified recombinant histidine-tagged (his)-IE1 from CMV was used asantigen coat. Briefly, BHK cells were transfected with VEE replicon RNAexpressing his-IE1 and Triton-X 100 lysates were prepared. Protein waspurified by ion metal affinity chromatography.

[0098] Sera from Day 51 (7 days post boost) were evaluated for thepresence of CMV-IE1-specific antibodies by a standard indirect ELISA.For detection of CMV-IE1-specific total Ig, a secondary polyclonalantibody that detects IgM, IgG and IgA was used for end point titerdetermination. Briefly, 96-well Maxisorp ELISA plates (Nunc, Naperville,Ill.) were coated with 2 μg IE1 in a volume of 50 μL incitrate/phosphate, pH 8.3, per well. Plates were covered with adhesiveplastic and incubated overnight at 4° C. The next day, unbound antigenwas discarded and plates were incubated for 1 hour with 200 μl blockingbuffer (PBS containing 3% w/v BSA) at room temperature. Wells werewashed 6 times with PBS and 50 μl of serum, diluted serially two-fold inbuffer (PBS with 1% w/v BSA and 0.05% v/v Tween 20), was added toantigen-coated wells. An α-IE1 monoclonal antibody (Rumbaugh-GoodwinInstitute for Cancer Research, Inc, Plantation, Fla.) was included inevery assay as a positive control. Negative controls in each assayincluded blanks (wells with all reagents and treatments except serum)and pre-bleed sera. Plates were incubated for one hour at roomtemperature, and then rinsed 6 times with PBS. Fifty μL/well of alkalinephosphatase (AP)-conjugated goat anti-mouse poly-isotype secondaryantibody (Sigma) diluted to a predetermined concentration in diluentbuffer was added to each well and incubated for 1 hour at roomtemperature. Wells were rinsed 6 times with PBS before addition of 100μl p-nitrophenyl phosphate (pNPP) substrate (Sigma). The serum antibodyELISA titer was defined as the inverse of the greatest serum dilutiongiving an optical density at 405 nm greater than or equal to 0.2 abovethe background (blank wells).

[0099] Summary of Immune Response to Multi-antigenic ARP

[0100] As shown in FIG. 1 and Table 3, animals vaccinated withmulti-antigenic ARP mounted immune responses to all seven antigenspresent in the ARP population. These immune responses included bothhumoral and cellular responses, indicating this type of approach canstimulate both arms of the immune system. The strength of the immuneresponse to a specific antigen was also measured in the context of themulti-antigenic ARP and compared to a single-antigen ARP preparation.Anti-Gag antibody and cellular immune responses were equivalent whetherthe HIV-Gag ARP was alone or in a multi-antigenic formulation,indicating that addition of a plurality of different antigens does notappear to diminish the immune response to each individual component ofthe preparation. This multi-antigenic preparation was intentionallycomposed of genes from infectious disease agents (HIV and CMV), cancerantigen (Rat/neu) and bacterial enzyme (CAT) to demonstrate that thehost immune system can be stimulated with multi-antigenic ARP to respondto a broad array of antigen types within a single ARP preparation.

Example 7 Animal Studies with Multi-Antigenic ARPs Expressing a TumorcDNA Library

[0101] A cDNA library is generated from a B16F10(B16) [Gold et al.,(2003) J. Immunol. 170:5188-5194) pigmented mouse melanoma cell lineoriginally derived from C576BU6 mice. This library is directionallycloned into the alphaviral replicon cDNA construct so that theheterologous cDNA is expressed from the replicon upon infection of atarget cell. ARP are generated and purified as described above toproduce a population of ARP particles expressing an entire library ofcDNAs from the B16 tumor cells. Expression of representative genes suchas β-actin can be analyzed by quantitative PCR to determine whether thelibrary expresses known gene standards. Subtractive hybridization ordifferential display against a non-tumorigenic genetically matched cellline can be used to enhance the proportion of tumor-specific sequencesin the library.

[0102] C57BL/6 mice are vaccinated with the B16 library ARP preparationone, two or three times on days 0, 21 and 42. Doses of between 10⁵-10⁹i.u. in ARP are administered via a subcutaneous (sc.) route deliveredboth rear footpads of the mouse. Control groups of mice receive placebovaccinations or ARP expressing irrelevant antigens. An additional set ofanimals can be included which receive ARP expressing single knownmelanoma specific tumor antigens such as TYR, TRP-2, gp100, MAGE-1 orMAGE-3, or a combination of said antigens as comparators to themulti-antigenic approach.

[0103] Mice are injected intradermally (id.) with 10⁴, 10⁵ or 10⁶ B16melanoma cells on the right flank 5 days after the final ARPimmunization. The mice are then followed for tumor onset by palpationevery other day. Tumors are scored as present once they reach a diameterof equal to or greater than 2 mm. Mice are sacrificed once it is assuredthat the tumor is progressing (usually at a size of 1 cm). Kaplan-Meiertumor-free survival curves are constructed and log rank analysisperformed to determine statistical significance of protection from tumorchallenge between each group.

[0104] Prior to tumor challenge, sera and lymphocytes are harvested frommice for immunoassay. The presence of humoral or cellular responses toknown tumor antigens expected to be present in the ARP B16 library canbe assayed using standard methods and techniques known in the art.

[0105] Canine malignant melanoma (CMM) is a spontaneous, aggressive andmetastatic neoplasm which occurs in dogs. CMM is a relatively frequentlydiagnosed tumor and accounts for about 4% of all canine tumors. CMM isinitially treated with local therapies including surgery and/orfractionated radiation therapy; however, systemic metastatic disease isa common sequela. CMM is a chemo-resistant neoplasm. All theseproperties are common to human melanoma, and on the basis of thesesimilarities, CMM serves as a clinical model for evaluating newtreatments for human melanoma [Bergman et al. (2003) Clin. Cancer Res.9:1284-1290).

[0106] Dogs are screened for the presence of histologically confirmedspontaneous malignant melanoma. Pre-trial evaluation includes completephysical evaluation, complete blood count and platelet count, serumchemistry profile, urinalysis, LDH, anti-nuclear antibody, andthree-dimensional measurements of the primary tumor if present (ormaximal tumor size from medical records if patient has been treatedbefore pretrial considerations). For the evaluation of metastaticdisease, 3-view radiographs of the thorax are obtained and regionallymph nodes are evaluated with fine needle aspiration/cytology and/orbiopsy/histopathology. All dogs are staged according to the WHO stagingsystem of stage II tumors (tumors 2-4 cm diameter, negative nodes),stage III (tumor >4 cm and/or positive nodes) or stage IV (distantmetastatic disease). Dogs from all three of these stages of disease areincluded in the study, provided they have not received any other form oftherapy in the previous three weeks.

[0107] Fine needle aspiration or biopsy is used to confirm malignantmelanoma in each animal by cytology or histopathology, respectively.These samples, taken from either the primary tumor mass or frommetastatic masses, are used as the source of the tumor cDNA library. Foreach animal, tumor RNA is isolated form the tumor cell population. AcDNA library is prepared from each sample. Multi-antigenic ARPpreparations are generated for each animal as described herein.

[0108] Cohorts of dogs receive multiple vaccinations of caninepatient-specific ARP preparations with a range of dosages. Dogs arevaccinated between 3-12 times over a period of 1-3 months. Dosages ofARPs administered via either a subcutaneous, intradermal orintramuscular route range from 10⁶ to 10⁹ i.u. In addition toadministering patient-specific (autologous) ARP vaccines, some cohortscan receive ARP preparations from other patients (allogeneic) in orderto determine if a vaccine preparation from an alternate melanomaprovides clinical benefit.

[0109] The clinical status of each patient is monitored throughout thevaccination regime and for up to two years following treatment. Patientsare physically, radiologically and biochemically examined on a frequentbasis for clinical evidence of tumor presence and progression orregression. If euthanasia is requested by owners in the event ofdegradation in the quality of life due to advanced disease, a fullnecropsy is performed with subsequent necropsy examination to determinegross and histopathological status of the tumor at primary andmetastatic sites. Statistical analysis is performed to determine theeffect of multi-antigenic ARP vaccination on survival and diseaseprogression. Statistical analysis tools include the Kaplan-Meier productlimit method, Cox proportional hazard analysis, Mann-Whitney U test, anda Spearman rank correlation. TABLE 1 Titration of Multi-antigenic ARPs(Pool of 10 constructs) Replicon vector ARP titer CAT(chloramphenicolacetyltransferase) 3.6 × 10⁸/mL β-gal 1.3 × 10⁵/mL Rat/neu 5.2 × 10⁸/mLLuciferase 6.8 × 10⁶/mL PkMSP1-42 4.5 × 10⁸/mL PyHep17 2.0 × 10⁸/mLPfAMA1 4.0 × 10⁷/mL PkCSP 5.7 × 10⁸/mL HIV Gag 1.5 × 10⁹/mL CancerAntigen A 4.5 × 10⁸/mL Total/ml 4.1 × 10⁹/mL Total from single cuvetteelectroporation 8.2 × 10¹⁰

[0110] TABLE 2 Titration of Multi-antigenic ARPs Produced from a Pool ofSeven RNAs ARP titer ARP titer in Replicon vector in media salt wash CMVIE1 2.9 × 10⁶/mL 1.9 × 10⁸/mL CMV gB 2.9 × 10⁵/mL 5.8 × 10⁷/mL InfluenzaHA 1.3 × 10⁵/mL 1.9 × 10⁷/mL HIV pol 3.4 × 10⁶/mL 3.3 × 10⁸/mL HIV Gag4.2 × 10⁷/mL 2.9 × 10⁹/mL Rat/neu 1.9 × 10⁶/mL 2.6 × 10⁸/mLCAT(chloramphenicol acetyltransferase) 2.3 × 10⁶/mL 2.9 × 10⁸/mLTotal/mL 5.7 × 10⁷/mL 4.1 × 10⁹/mL Total from single cuvette 1.1 × 10¹⁰8.2 × 10¹⁰ electroporation Total Pooled ARP Titer 9.3 × 10¹⁰

[0111] TABLE 3 Antigen-specific Immune Responses in Animals Immunizedwith Multi-antigenic ARPs Vaccination HIV GAG HIV GAG HIV POL FLU HA CMVgB CAT Rat/neu CMV IE1 Group ELISA ELISPOT ELISPOT IFA Western ELISAELISA ELISA Multi-Ag ARP  8192 475 614 160^(a) 80^(a) 1280^(a) 1280^(a)1280 s.c. footpad Multi-Ag ARP 40960^(a) 439 105 160^(a) 80^(a) 1280^(a) 320 2560^(a) i.p. HIV GAG ARP  5120 500  0^(b)  10^(b) 10^(b)  10^(b) 10^(b)  10^(b) s.c. footpad Negative  10^(b)  0^(b)  0^(b)  10^(b)10^(b)  10^(b)  10^(b)  10^(b) control s.c. footpad

[0112] References Cited in the Present Application

[0113] Casimiro D R, Tang A, Perry H C, Long R S, Chen M, Heidecker G J,Davies M E, Freed D C, Persaud N V, Dubey S, Smith J G, Havlir D,Richman D, Chastain M A, Simon A J, Fu T M, Emini E A, Shiver J W.Vaccine-induced immune responses in rodents and nonhuman primates by useof a humanized human immunodeficiency virus type 1 pol gene. J.Virology. 2002. 76:185-195, 2002

[0114] Chen G J, Qiu N, Karrer C, Caspers P, and Page M G. Restrictionsite-free insertion of PCR products directionally into vectors.Biotechniques. 2000; 28(3):498-500, 504-5.

[0115] Chen G J, Qiu N, Page M P. Universal restriction site-freecloning method using chimeric primers. Biotechniques. 2002; 32(3):516,518-20.

[0116] Heiser, A., Coleman, D., Dannull, J., Yancy, D., Maurice, M.,Lallas, C., Dahm, P., Niedzwiecki, D., Gilboa, E. and J. Vieweg. J.Clinical Investigation. 2002. 109(3):409-417.

[0117] Kumamoto, T., Huang, E., Paek, H-J., Morita, A., Matsue, H.,Valentini, R., and A. Takashima. Nature Biotechnology. 2002. 20:64-69.

[0118] Rayner, J O, Dryga, S. A. and Kurt I. Kamrud. Alphavirus vectorsand vaccination. Rev. Med. Virol. 2002. 12:279-296.

[0119] Sadanaga N, Nagashima H, Mashino K, Tahara K, Yamaguchi H, OhtaM, Fujie T, Tanaka F, Inoue H, Takesako K, Akiyoshi T, Mori M. Dendriticcell vaccination with MAGE peptide is a novel therapeutic approach forgastrointestinal carcinomas. Clin. Cancer Res. 2001 August;7(8):2277-84.

[0120] Yamanaka, R., Zullo, S. A., Tanaka, R., Blaese, M., and K. G.Xanthopoulos. Enhancement of antitumor immune response in glioma modelsin mice by genetically modified dendritic cells pulsed with Semlikiforest virus-mediated complementary DNA. J. Neurosurg. 2001.94(3):474-81.

[0121] Ward S, Casey D, Labarthe M C, Whelan M, Dalgleish A, Pandha H,Todryk S. Immunotherapeutic potential of whole tumour cells. CancerImmunol. Immunother. 2002. 51(7):351-7.

[0122] Ying, H., Zaks, T. Z. Rong-Fu, W., Irvine, K. R., Kammula, U. S.Marincola, F. M. Leiner, W. W. and N. P. Restifo. Cancer therapy using aself-replicating RNA vaccine. Nature Medicine. 1999. 7(5):823-827

1 3 1 11 PRT Artificial Sequence peptide epitope of murine Pol CTL 1 ValTyr Tyr Asp Pro Ser Lys Asp Leu Ile Ala 1 5 10 2 11 PRT ArtificialSequence Peptide epitope of murine Pol CTL 2 Glu Leu Arg Gln His Leu LeuArg Trp Gly Leu 1 5 10 3 11 PRT Artificial Sequence Peptide epitope ofmurine Pol CTL 3 Glu Leu Arg Glu His Leu Leu Lys Trp Gly Phe 1 5 10

What is claimed is:
 1. A method for preparing alphaviral repliconparticles (ARPS) encoding and expressing a plurality of antigens, saidmethod comprising the steps of: a) introducing a plurality of alphaviralreplicon nucleic acids into a plurality of cells, wherein said cells arepermissive for alphavirus replication and packaging, wherein saidreplicon nucleic acid comprises at least a virus packaging signal and atleast one heterologous coding sequence expressible in said alphaviralreplicon nucleic acid, wherein said cell comprises at least one helperfunction, to produce a modified cell, and wherein the plurality ofalphaviral replicon nucleic acids encode a plurality of antigens, toproduce a plurality of modified cells; b) culturing said plurality ofmodified cells of step (a) under conditions allowing expression of theat least one helper function, allowing replication of said alphaviralreplicon nucleic acid and packaging of said alphaviral replicon nucleicacid to form ARPs; c) contacting the modified cells after step (b) withan aqueous solution having an ionic strength from 0.2M to 5M to releasethe ARPs into the aqueous solution to produce a ARP-containing solution;and d) collecting ARPs from the ARP-containing solution of step (c). 2.The method of claim 1 wherein the at least one helper function in thehost cell of step (a) is encoded by a nucleic acid sequence stablyintegrated within the genome of said host cell.
 3. The method of claim 1wherein the at least one helper function in the cell is introduced on atleast one helper nucleic acid which encodes a capsid protein capable ofbinding said alphaviral replicon nucleic acid, and at least onealphaviral glycoprotein, wherein said alphaviral glycoprotein associateswith said alphaviral replicon nucleic acid and said capsid protein,wherein the at least one helper nucleic acid molecule is introduced intothe cell together with said alphaviral replicon nucleic acid.
 4. Themethod of claim 1, wherein the at least one helper function is encodedby at least two helper nucleic acid molecules wherein each of said twohelper nucleic acid molecules encodes at least one alphaviral helperfunction.
 5. The method of claim 1, wherein the at least one helpernucleic acid molecule and the alphaviral replicon RNA are RNA molecules.6. The method of claim 5, wherein the at least one helper nucleic acidmolecule is not capped.
 7. The method of claim 1, wherein at least onehelper nucleic acid molecule is a DNA molecule.
 8. The method of claim1, wherein the replicon nucleic acid is introduced into said host cellby electroporation.
 9. The method of claim 8, wherein the cell densityin the electroporation milieu is from 10⁷ to 5×10⁸ per mL.
 10. Themethod of claim 8, wherein the electroporation is carried out in anelectroporation cuvette.
 11. The method of claim 1, wherein step (d) isfollowed by an ion exchange chromatography step or a heparin affinitychromatography step.
 12. The method of claim 1, wherein the alphavirusis an attenuated alphavirus.
 13. The method of claim 12, wherein theattenuated alphavirus is Venezuelan equine encephalitis virus (VEE). 14.The method of claim 13, wherein the attenuated VEE is strain
 3014. 15.The method of claim 1, wherein the wash step employs NaCl, KCl, MgCl₂,CaCl₂, NH₄Cl, (NH₄)₂SO4, NH₄ Acetate or NH₄ Bicarbonate.
 16. Analphavirus replicon particle preparation prepared by the method ofclaim
 1. 17. The alphavirus replicon particle preparation of claim 16,wherein the plurality of encoded antigens are derived from tumor cells.18. The alphavirus replicon particle preparation of claim 16, whereinthe plurality of encoded antigens are derived from a parasite or apathogen.
 19. The alphavirus replicon particle preparation of claim 18,wherein the plurality of encoded antigens are derived from a pathogenselected from the group consisting of viruses, fungi, yeasts, bacteriaand protozoans.
 20. A method for immunizing a human or animal against aparasite, pathogen or cancer, said method comprising the step ofadministering an amount of a virus replicon particle preparation ofclaim 15 effective for generating an immune response to at least oneantigen of said parasite, pathogen or cancer.
 21. The method of claim20, wherein the pathogen is a virus, a bacterium, a yeast, a fungus or aprotozoan.
 22. The method of claim 21, wherein the virus is an influenzavirus, a herpes virus, a parainfluenza virus, respiratory syncytialvirus, cytomegalovirus, human papilloma, or human immunodeficiencyvirus.
 23. The method of claim 21, wherein the protozoan is Plasmodiumfalciparum.
 24. The method of claim 21, wherein the bacterium isMycobacterium tuberculosis.
 25. The method of claim 20, wherein thecancer is selected from the group consisting of pancreatic cancer,kidney cancer, sarcoma, neuroblastoma, glioma, colon cancer, melanoma,breast cancer, ovarian cancer and prostate cancer.
 26. A method forpreparing alphaviral replicon particles (ARPs) encoding and expressing aplurality of antigens, said method comprising the steps of: a)introducing a plurality of alphaviral replicon nucleic acids into aplurality of cells, wherein said cells are permissive for alphavirusreplication and packaging, wherein said replicon nucleic acid comprisesat least a virus packaging signal and at least one heterologous codingsequence expressible in said alphaviral replicon nucleic acid, whereinsaid cell comprises at least one helper function, to produce a modifiedcell, and wherein the plurality of alphaviral replicon nucleic acidsencode a plurality of antigens, to produce a plurality of modifiedcells, wherein the step of introducing the nucleic acids is byelectroporating said cells at a density from 5×10⁷ to 5×10⁸ per mL ofelectroporation mixture; b) culturing said plurality of modified cellsof step (a) under conditions allowing expression of the at least onehelper function, allowing replication of said alphaviral repliconnucleic acid and packaging of said alphaviral replicon nucleic acid toform ARPs; c) contacting the modified cells after step (b) with anaqueous solution having an ionic strength from 0.2M to 5M to release theARPs into the aqueous solution to produce a ARP-containing solution; andd) collecting ARPs from the ARP-containing solution of step (c).